Fibroblast growth factor mutants having improved functional half-life and methods of their use

ABSTRACT

Mutant fibroblast growth factor (FGF) proteins having a polypeptide sequence with a high sequence identity to proteins encoded by members of the Fgf-1 subfamily of genes from a mammalian species, such as human, and with a specific amino acid substitution of an alanine at a position corresponding to amino acid position 66 of human FGF-1 with a cysteine and/or a specific amino acid substitution of a phenylalanine at a position corresponding to amino acid position 132 of human FGF-1 with a tryptophan (based on the 140 amino acid numbering scheme of human FGF-1) are provided. Other amino acid mutations or substitutions may be combined. Polynucleotide sequences encoding the mutant FGF proteins and host cells containing such polynucleotide sequences are provided. Methods of administering a mutant FGF protein to an individual to treat an ischemic condition or disease or a wound or tissue injury are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/295,833,filed Oct. 17, 2016, which is a continuation of U.S. patent applicationSer. No. 14/593,107, filed Jan. 9, 2015, which is a divisionalapplication of U.S. patent application Ser. No. 13/669,856 filed Nov. 6,2012, entitled “FIBROBLAST GROWTH FACTOR MUTANTS HAVING IMPROVEDFUNCTIONAL HALF-LIFE AND METHODS OF THEIR USE, which is a divisionalapplication of U.S. patent application Ser. No. 12/783,005 filed May 19,2010, now U.S. Pat. No. 8,461,111, entitled “FIBROBLAST GROWTH FACTORMUTANTS HAVING IMPROVED FUNCTIONAL HALF-LIFE AND METHODS OF THEIR USE,”which claims the benefit of priority to U.S. Provisional App. No.61/179,751 filed May 20, 2009 entitled “FIBROBLAST GROWTH FACTOR MUTANTSHAVING IMPROVED FUNCTIONAL HALF-LIFE AND METHODS OF THEIR USE,” and U.S.Provisional App. No. 61/309,590 filed Mar. 2, 2010 entitled same, thecontents and disclosures of which are hereby incorporated by referencein their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 2, 2012, isnamed 74606101.txt and is 19,710 bytes in size.

FIELD OF THE INVENTION

The present invention broadly relates to mutant fibroblast growth factor(FGF) proteins having higher thermostability and/or functional half-lifefor therapeutic applications. The present invention also relates topolynucleotides encoding such mutant FGF proteins and cells that may beused for expression. The present invention further broadly relates tomethods of administering mutant FGF proteins to an individual, such asan individual having an ischemic or hypoxic condition or in need ofwound healing or tissue repair.

BACKGROUND

Fibroblast growth factor-1 (FGF-1) is a potent mitogen and angiogenicfactor suggested for use as a protein biopharmaceutical in treating awide range of diseases and conditions. However, FGF-1 has poor thermalstability and a short half-life, and denatured or unfolded FGF-1 mayform aggregates and become immunogenic. Pharmaceutical compositions ofFGF-1 have been formulated with heparin to increase the stability andhalf-life of FGF-1. However, heparin has its own pharmacologicalproperties, which may complicate using heparin-containing formulationsof FGF-1. Therefore, a need continues in the art for new and improvedcompositions and methods for using modified versions of FGF-1 or relatedproteins having higher stability and longer functional half-life thatare non-immunogenic and avoid the need for heparin.

SUMMARY

According to a first broad aspect of the present invention, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 90% identical to the polypeptide sequence ofwild-type human FGF-1 protein (SEQ ID NO. 1), wherein the alanine (Ala)at an amino acid position of the mutant FGF protein corresponding toamino acid position 66 of wild-type human FGF-1 is replaced withcysteine (Cys), wherein the numbering of the amino acid positions isbased on the numbering scheme for the 140 amino acid form of humanFGF-1.

According to a second broad aspect of the present invention, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 90% identical to the polypeptide sequence ofwild-type human FGF-1 protein (SEQ ID NO. 1), wherein the phenylalanine(Phe) at an amino acid position of the mutant FGF protein correspondingto amino acid position 132 of wild-type human FGF-1 is replaced withtryptophan (Trp), wherein the numbering of the amino acid positions isbased on the numbering scheme for the 140 amino acid form of humanFGF-1.

According to a third broad aspect of the present invention, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 90% identical to the polypeptide sequence ofwild-type human FGF-2 protein (SEQ ID NO: 4), wherein the alanine (Ala)at an amino acid position of the mutant FGF protein corresponding toamino acid position 84 of wild-type human FGF-2 is replaced withcysteine (Cys).

According to a fourth broad aspect of the present invention, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 90% identical to the polypeptide sequence ofwild-type human FGF-2 protein (SEQ ID NO: 4), wherein the phenylalanine(Phe) at an amino acid position of the mutant FGF protein correspondingto amino acid position 148 of wild-type human FGF-2 is replaced withtryptophan (Trp).

According to a fifth broad aspect of the present invention,polynucleotide sequences encoding a mutant FGF protein of the presentinvention and host cells containing such polynucleotide sequences areprovided.

According to a sixth broad aspect of the present invention, apharmaceutical composition comprising a mutant FGF protein of thepresent invention and a pharmaceutically acceptable carrier is provided.

According to a seventh broad aspect of the present invention, a methodis provided comprising the following steps: (a) identifying anindividual having an ischemic condition or disease; and (b)administering to the individual a composition, comprising a mutant FGFprotein of the present invention.

According to a eighth broad aspect of the present invention, a method isprovided comprising the following steps: (a) identifying an individualhaving a wound or tissue damage; and (b) administering to the individuala composition comprising a mutant FGF protein of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will be described in conjunctionwith the accompanying drawings, in which:

FIG. 1 is the polypeptide sequence of the 155 amino acid form ofwild-type human FGF-1 protein (SEQ ID NO: 2) with boxes showing alanine(A) at position 81 and phenylalanine (F) at position 147 that may eachbe mutated to or substituted with cysteine (C) and tryptophan (W),respectively, for mutant FGF proteins of the present invention. Shadedresidues are removed (i.e., not present) in the 140 amino acid form ofhuman FGF-1, and the underlined methionine (M) is removed (i.e., notpresent) in the 154 amino acid form of human FGF-1.

FIG. 2 is the polypeptide sequence of the 140 amino acid form ofwild-type human FGF-1 protein (SEQ ID NO: 1) with boxes showing alanine(A) at position 66 and phenylalanine (F) at position 132 that may eachbe mutated to or substituted with cysteine (C) and tryptophan (W),respectively, for mutant FGF proteins of the present invention.

FIG. 3 is the polypeptide sequence of the 155 amino acid form ofwild-type mouse FGF-1 protein (SEQ ID NO: 11) with boxes showing alanine(A) at position 81 and phenylalanine (F) at position 147 that may eachbe mutated or substituted to cysteine (C) and tryptophan (W),respectively, for mutant FGF proteins of the present invention.

FIG. 4 is the polypeptide sequence of the 155 amino acid form ofwild-type human FGF-2 protein (SEQ ID NO: 4) with boxes showing alanine(A) at position 84 and phenylalanine (F) at position 148 that may eachbe mutated to or substituted with cysteine (C) and tryptophan (W),respectively, for mutant FGF proteins of the present invention.

FIG. 5 is the polypeptide sequence of the 154 amino acid form ofwild-type mouse FGF-2 protein (SEQ ID NO: 12) with boxes showing alanine(A) at position 83 and phenylalanine (F) at position 147 that may eachbe mutated to or substituted with cysteine (C) and tryptophan (W),respectively, for mutant FGF proteins of the present invention.

FIG. 6 is a table showing the thermodynamic parameters for wild-type andmutant FGF-1 proteins in a crystallization buffer as determined byisothermal equilibrium denaturation using GuHCl and monitored usingfluorescence spectroscopy.

FIG. 7-1 is a table showing crystallographic data collection andrefinement statistics for mutant FGF-1 proteins. FIG. 7-2 is acontinuation of the table shown in FIG. 7-1.

FIG. 8 is graph showing isothermal equilibrium denaturation profiles forthe Ala66→Cys mutant with air oxidation for the indicated time periodswith the time-dependent conversion of the reduced form to the oxidizedform associated with significantly enhanced stability.

FIG. 9 is a graph showing a progress curve for the fractional increasein the oxidized form of the Ala66→Cys mutant protein from the isothermalequilibrium data in FIG. 8 with the fitted function having a first orderexponential fit and yielding a first-order rate constant (i.e.,half-life) of about 60 hours. An inset of a Coomassie blue-stained SDSPAGE gel is shown with mass standards in lane 1, a reduced wild-typeFGF-1 in lane 2, a non-reduced wild-type FGF-1 in lane 3, a reducedAla66→Cys FGF-1 mutant incubated in air oxidizing conditions for 48hours in lane 4, and a non-reduced Ala66→Cys FGF-1 mutant incubated inair oxidizing conditions for 48 hours in lane 5 (reduced samples exposedto DTT prior to gel loading).

FIG. 10 is a graph showing the normalized fractional denaturationprofiles for reduced and oxidized forms of the Ala66→Cys mutant FGF-1protein and overlaid with the profile for wild-type FGF-1 with thedenaturation profile for the oxidized form of the Ala66→Cys mutant FGF-1protein obtained from a global fit to the oxidized component in thedenaturation profiles shown in FIG. 8.

FIGS. 11A, 11B, 11C, 11D and 11E are a set of stereo diagrams of X-raycrystal structures (molecule A in the asymmetric unit in each case)showing the structural details of mutant FGF-1 adjacent to the site ofmutation at position 83 mutants of FGF-1 (CPK coloring) overlaid withthe coordinates of wild-type FGF-1 (1JQZ; grey coloring) with theCys83→Ala mutation (FIG. 11A), the Cys83→Ser mutation (FIG. 11B), theCys83→Thr mutation (FIG. 11C), the Cys83→Val mutation (FIG. 11D), andthe Cys83→Ile mutation (FIG. 11E) shown. FIGS. 11A, 11B, 11C, 11D and11E disclose “NEEA,” “NEES,” “NEET,” “NEEV,” and “NEEI” as SEQ ID NOS15-19, respectively.

FIG. 12 is a relaxed stereo diagram of the X-ray crystal structures ofthe reduced and oxidized forms of the Ala66→Cys mutant, (top): moleculeA in the asymmetric unit of reduced Ala66→Cys overlaid with wild-typeFGF-1 (1JQZ; light gray), (middle): molecule B in the asymmetric unit ofreduced Ala66→Cys overlaid with wild-type FGF-1 (1JQZ; light gray),(bottom): molecule A of oxidized Ala66→Cys overlaid with wild-type FGF-1(1JQZ; light gray).

FIG. 13-1 is a table showing crystallographic data collection andrefinement statistics for mutant FGF-1 proteins. FIG. 13-2 is acontinuation of the table shown in FIG. 13-1.

FIG. 14 is a table showing thermodynamic parameters for FGF-1 mutantsdetermined from isothermal equilibrium denaturation using GuHCl in ADAbuffer.

FIG. 15 is a table showing DSC data in ADA buffer in the presence of0.7M GuHCl.

FIG. 16 is a table showing mitogenic activity of wild-type and mutantFGF-1 proteins in the absence and presence of 10 U/ml heparin on NIH 3T3fibroblasts, protein functional half-life in unconditioned medium, andprotein half-life with 200:1 trypsin digestion.

FIG. 17 is a table showing the mitogenic activity and functionalhalf-life of the wild-type FGF-1 and Ala66→Cys (oxidized form) mutant.

FIG. 18 is a relaxed stereo ribbon diagram of wild-type FGF-1 (1JQZ;molecule A) indicating the location of the eight solvent-excludedcavities identified using a 1.2 Å radius probe with residues borderingthe cavities indicated in single letter amino acid code, and underlinedresidues having solvent accessibility.

FIGS. 19A, 19B, 19C, 19D and 19E are a set of relaxed stereo diagrams ofthe Leu44→Trp mutant (FIG. 19A), Phe85→Trp mutant (FIG. 19B), Phe132→Trpmutant (FIG. 19C), Val31→Ile mutant (FIG. 19D), and Cys117→Ile mutant(FIG. 19E) with each overlaid onto the wild-type FGF-1 (1JQZ) structure(dark gray) and with the cavities adjacent to these mutant positionswithin the wild-type structure that are detectable using a 1.2 Å radiusprobe shown.

FIGS. 20A and 20B are a set of relaxed stereo diagrams of theLeu44→Phe+Phe132→Trp double mutant of FGF-1 FIG. A, and theLeu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp quadruple mutant of FGF-1 FIG.B overlaid onto the wild-type (1JQZ) FGF-1 structure (dark gray) withFIG. A showing the location of the three cavities (cav4, cav5 and cav6)that are filled in response to the Leu44→Phe+Phe132→Trp double mutationand with FIG. B the overlaid with the wild-type structure and showingonly the main chain atoms within 5 Å of positions 44, 83, 117, and 132.

FIG. 21 is a set of graphs of a NIH 3T3 fibroblast mitogenic assay ofwild-type and mutant forms of FGF-1 in the absence (top) and presence(bottom) of 10 U/ml heparin.

FIGS. 22A and 22B are a set of graphs showing mitogenic activity ofwild-type FGF-1 (filled circles) and Ala66→Cys mutant (open circles) inthe absence (FIG. 22A) or presence (FIG. 22B) of 10 U/ml heparin.

FIG. 23 is a graph showing the log of the percent initial mitogenicactivity plotted as a function of incubation time (i.e., functionalhalf-life) prior to measurement of mitogenic activity providinginactivation rates of wild-type and mutant forms of FGF-1 inunconditioned DMEM at 37° C.

FIG. 24 is a graph showing the functional half-life assay for wild-typeFGF-1 (filled circles) and Ala66→Cys mutant (open circles).

FIGS. 25A, 25B, 25C, 25D and 25E are a set of images of CoomassieBrilliant Blue stained 16.5% Tricine SDS-PAGE gels of a time-courseincubation in TBS of wild-type FGF-1 (FIG. 25A), Cys117→Val mutant FGF-1(FIG. 25B), Cys83→Thr+Cys117→Val mutant FGF-1 (FIG. 25C),Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp mutant FGF-1 (FIG. 25D), andLys12→Val+Cys83→Thr+Cys117→Val (FIG. 25E) with samples labeled “reduced”made in 5% BME prior to gel loading.

FIG. 26 is a time course plot of the proteolytic digestion of thewild-type and mutant forms of FGF-1 by trypsin (200:1 molar ratio,respectively) in TBS pH 7.4 at 37° C. showing the fraction of intactprotein quantified by scanning densitometry of Coomassie Blue stainedTricine SDS PAGE gels.

FIG. 27 is a relaxed stereo diagram showing an overlay of the main chainatoms of the Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp mutant (lightgray) with wild-type FGF-1 (dark gray) with this set of atoms overlaidwith a root-mean-square deviation of 0.25 Å and with conserved solventmolecules shown as spherical representations.

FIG. 28 is the polypeptide sequence of the 140 amino acid form ofwild-type human FGF-1 protein with boxes showing cysteine (C) atposition 83, cysteine (C) at position 117, leucine (L) at position 44and phenylalanine (F) at position 132 that are each mutated to orsubstituted with threonine (T), valine (V), phenylalanine (F) andtryptophan (W), respectively, for mutant FGF proteins of the presentinvention (SEQ ID NO: 13).

DETAILED DESCRIPTION Definitions

Where the definition of terms departs from the commonly used meaning ofthe term, applicant intends to utilize the definitions provided below,unless specifically indicated.

For purposes of the present invention, the terms “thermodynamicstability” or “thermostability” of a protein refer interchangeably tothe ability of a protein to maintain its tertiary structure (i.e., toresist denaturation or unfolding) at a given temperature or in thepresence of a denaturant. The thermostability of a protein (e.g., amutant FGF protein) may be expressed in terms of the Gibb's free energyequation relative to a standard (e.g., wild-type FGF protein) accordingto known methods.

For purposes of the present invention, the term “functional half-life”of a FGF protein refers to the amount of time it takes for the activityor effect of a FGF protein (e.g., a mutant FGF protein) to becomereduced by half. For example, the functional half-life may be based onthe activity of a FGF protein over time in inducing growth,proliferation, and/or survival of cells, such as according to a culturedfibroblast proliferation assay. The functional half-life of a proteinmay be different than the thermostability of the same protein sincethese are separable properties of proteins. For example, a protein maybe mutated such that the thermostability of the protein is decreasedwhile its functional half-life is increased.

For purposes of the present invention, the terms “correspond” or“corresponding” in reference to a protein sequence refer interchangeablyto an amino acid position(s) of a protein, such as a mutant FGF protein,that is equivalent or corresponds to an amino acid positions) of one ormore other protein(s), such as a wild-type FGF protein, according to anystandard criteria known in the art. An amino acid at a position of aprotein may be found to be equivalent or corresponding to an amino acidat a position of one or more other protein(s) based on any relevantevidence, such as the primary sequence context of the each amino acid,its position in relation to the N-terminal and C-terminal ends of itsrespective protein, the structural and functional roles of each aminoacid in its respective protein, etc. For proteins having similar ornearly identical polypeptide sequences, a “corresponding” amino acid(s)and corresponding amino acid position(s) between proteins may bedetermined or deduced by sequence alignment and comparison. However,“corresponding” amino acid(s) for two or more proteins may havedifferent amino acid position numbers or numbering (e.g., when countedfrom the N-terminus of each protein) since the two or more proteins mayhave different lengths and/or one or more substitutions, insertions,deletions, etc. For example, related proteins may have deletions orinsertions in relation to each other that offset the numbering of theirrespective or corresponding amino acid sequences (i.e., based on theirprimary structure or sequence). An amino acid position(s) of a protein,such as a mutant FGF protein, may “correspond” to an amino acidposition(s) of one or more other protein(s) if the amino acid positionsare structurally equivalent or similar when comparing thethree-dimensional structures (i.e., tertiary structures) of therespective proteins. A person skilled in the art would be able todetermine “corresponding” amino acids and/or “corresponding” amino acidpositions of two or more proteins based on their protein sequencesand/or protein folding or tertiary structure.

For purposes of the present invention, the terms “identical” or“identity” refer to the percentage of amino acid residues of two or morepolypeptide sequences having the same amino acid at correspondingpositions. For example, a protein that is at least 90% identical to apolypeptide sequence will have at least 90% of its residues that are thesame as those in the polypeptide sequence at corresponding positions.

For purposes of the present invention, the term “Fgf-1 subfamily” refersto the subfamily of mammalian genes, and mutants thereof, that are morerelated in sequence to mammalian Fgf-1 genes than other mammalian Fgfsubfamilies. For example, Fgf-1 subfamily members may include mammalianorthologs of Fgf-1 and Fgf-2. In addition, the term “FGF-1 subfamily” or“FGF A subfamily” refer to the subfamily of mammalian proteins, andmutants thereof, that are more related in sequence to mammalian FGF-1proteins than other mammalian FGF subfamilies. For example, FGF-1subfamily members may include mammalian FGF-1 and FGF-2.

For purposes of the present invention, the terms “individual,”“subject,” or “patient” refer interchangeably to a mammalian organism,such as a human, mouse, etc., that is administered a mutant FGF proteinof the present invention for a therapeutic or experimental purpose.

DESCRIPTION

Protein biopharmaceuticals are an important and growing area of humantherapeutics, but the intrinsic property of proteins to adoptalternative conformations (e.g., during protein unfolding) presentsnumerous challenges limiting their effective application asbiopharmaceuticals. Although still a comparatively small percentageoverall, protein biopharmaceuticals are the fastest-growing category ofnew drug approvals and currently target over 200 human diseases,including cancers, heart disease, Alzheimer's, diabetes, multiplesclerosis, AIDS, and arthritis. See, e.g., Crommelin, D. J. A., et al.,“Shifting paradigms: biopharmaceuticals versus low molecular weightdrugs,” International Journal of Pharmaceutics 266:3-16 (2003), theentire contents and disclosure of which are hereby incorporated byreference. The impact of protein biopharmaceuticals upon U.S. healthcareand the economy is substantial and growing rapidly. However, incomparison to traditional small molecules, proteins present new andsignificant challenges that need to be overcome before their fullpotential as therapeutic agents may be realized. One unique property ofproteins is that they are able to adopt different structuralconformations, and this profoundly influences critically-importantproperties of proteins, such as their function, solubility,bioavailability, half-life, aggregation, toxicity, immunogenicity, etc.See, e.g., Frokjaer, S. et al., “Protein drug stability: a formulationchallenge,” Nature Reviews 4:298-306 (2005); Hermeling, S. et al.,“Structure-immunogenicity relationships of therapeutic proteins,”Pharmaceutical Research 21:897-903 (2004); and Krishnamurthy, R. et al.,“The stability factor: importance in formulation development,” CurrentPharmaceutical Biotechnology 3:361-371 (2002), the entire contents anddisclosures of which are hereby incorporated by reference. A keyintrinsic property of proteins in this regard is their thermodynamicstability (ΔG_(unfolding)) which defines an equilibrium between nativeand denatured states of a protein.

The thermodynamic stability of a protein may be of particularsignificance in therapeutic applications because unfolded or aggregatedforms of a protein may be potentially toxic and/or immunogenic. Forexample, neutralizing antibodies in patients treated withinterferon-alpha 2a were observed when the protein is stored at roomtemperature and formed detectable aggregates, but both the formation ofaggregates and immunogenicity were reduced upon storage at 4° C. (whereΔG_(unfolding) increased). See, e.g., Hochuli, E. “Interferonimmunogenicity: technical evaluation of interferon-alpha 2a,” Journal ofInterferon and Cytokine Research 17:S15-S21 (1997), the entire contentsand disclosure of which are hereby incorporated by reference. In adifferent study, persistent antibodies were generated in patientstreated with human growth hormone with formulations containing 50-70%aggregates. However, when the formulation of human growth hormone wasmodified to contain less than 5% aggregates, only transient or noantibodies were observed. See, e.g., Moore, W. V. et al., “Role ofaggregated human growth hormone (hGH) in development of antibodies tohGH,” Journal of Clinical Endocrinology and Metabolism 51:691-697(1980), the entire contents and disclosure of which are herebyincorporated by reference. In yet another study using recombinantclotting factor VIII in mice, the formation of aggregates was associatedwith the emergence of entirely novel immunogenic epitopes. See, e.g.,Purohit, V. S. et al., “Influence of aggregation on immunogenicity ofrecombinant human Factor VIII in hemophilia A mice,” Journal ofPharmaceutical Sciences 95:358-371 (2006), the entire contents anddisclosure of which are hereby incorporated by reference. Thus, proteinstability, denaturation, aggregation and immunogenicity may be criticaland interrelated issues influencing the successful application ofproteins as biopharmaceuticals.

Various efforts have been made to increase the thermodynamic stabilityand/or half-life of proteins that are intended for use asbiopharmaceuticals, while reducing their aggregation and/orimmunogenicity. One such approach uses covalent attachment ofpolyethylene glycol (PEG), a highly soluble and biocompatible polymer,to substantially increase the circulating half-life of proteins throughreduced renal clearance due to a substantial increase in the molecularmass of these proteins. The attached PEG molecule may also physicallymask regions of the protein that would otherwise be susceptible toproteolytic attack or immune recognition, increasing further thecirculating half-life and reducing immunogenicity. However, attachmentof PEG molecules (“PEGylation”) typically does not increase the formalthermodynamic stability of proteins and has been noted to reduce thethermodynamic stability in some cases. See, e.g., Basu, A., et al.,“Structure-function engineering of interferon-β-1b for improvingstability, solubility, potency, immunogenicity, and pharmacokineticproperties by site-selective mono-PEGylation,” Bioconjugate Chemistry 17(2006); and Monfardini, C. et al., “A branched monomethoxypoly(ethyleneglycol) for protein modification,” Bioconjugate Chemistry 6:62-69(1995), the entire contents and disclosure of which are herebyincorporated by reference.

Therefore, the beneficial properties of PEGylation are primarilyassociated with modulation of renal clearance and a reduction inproteolysis and immune recognition (i.e., PEGylation generally docs notincrease thermodynamic stability of a protein). One problem withPEGylation is that it may interfere with critical functional interfaceson the protein surface, often reducing receptor/ligand affinity by twoor more orders of magnitude. However, PEGylation studies do show thatshielding of epitopes on the protein surface may substantially reduce oreliminate the immunogenic potential of a protein, which may haveimportant ramifications for protein engineering by suggesting thatmutations at solvent-inaccessible positions within proteins may minimizetheir immunogenic potential.

The fibroblast growth factors (gene=Fgf; protein=FGF) are a family ofpolypeptides with diverse roles in development and metabolism. Fgfs havebeen found in many multicellular organisms, ranging from Caenorhabditiselegans to Homo sapiens. Two Fgf genes have been identified in C.elegans, while the mouse and human each share 22 Fgf genes. See, e.g.,Itoh, N. et al., “Evolution of the Fgf and Fgfr gene families,” TrendsGenet 20 (2004); Popovici, C. et al., “An evolutionary history of theFGF superfamily,” BioEssays 27:849-857 (2005); and Itoh, N. et al.,“Functional evolutionary history of the mouse Fgf gene family,”Developmental Dynamics 237:18-27 (2008), the entire contents anddisclosures of which are hereby incorporated by reference. Fgf genesgenerally encode potent mitogens for a broad spectrum of cell types,including vascular cells.

Human and mouse Fgf genes and proteins may be divided into sevensubfamilies based on phylogenetic analysis: Fgf-1 or FGF A subfamily(including FGF-1 and FGF-2 proteins); Fgf-4 or FGF C subfamily(including FGF-4, FGF-5, and FGF-6 proteins); Fgf-7 or FGF B subfamily(including FGF-3, FGF-7, FGF-10, and FGF-22 proteins); Fgf-8 or FGF Dsubfamily (including FGF-8, FGF-17, and FGF-18 proteins); Fgf-9 or FGF Esubfamily (including FGF-9, FGF-16, and FGF-20 proteins); intracellulariFgf or FGF F subfamily (including FGF-11, FGF-12, FGF-13, and FGF-14proteins); and hormone-like hFgf or FGF G subfamily (includingFGF-15/FGF-19, FGF-21, and FGF-23 proteins). See, e.g., Itoh, N. et al.(2008), supra; Popovici, C. et al. (2005), supra; and Ornitz, D. M. etal., “Fibroblast growth factors,” Genome Biology 2(3):3005.1-3005.12(2001), the entire contents and disclosure of which are herebyincorporated by reference.

Several members of the FGF family of proteins, including FGF-1 subfamilyFGF-1 and FGF-2 proteins (also referred to as acidic FGF and basic FGF,respectively), have the potential of providing “angiogenic therapy” forthe treatment of ischemic conditions or diseases (i.e., diseases causedby insufficient blood flow to one or more tissues), such as coronaryartery disease, peripheral vascular disease, peripheral arterialocclusion or disease (e.g., critical limb ischemia or CLI), etc., bytriggering neovascularization of affected tissues. See, e.g., Nikol, S.et al., “Therapeutic Angiogenesis With Intramuscular NVIFGF ImprovesAmputation-free Survival in Patients With Critical Limb Ischemia,” MolTher 16(5):972-978 (2008), the entire contents and disclosure of whichare hereby incorporated by reference. In addition, FGF proteins may beused for tissue repair and wound healing by triggering angiogenesis andproliferation of fibroblasts involved in healing damaged tissue andfilling the wound space with new tissue.

FGF-1 has also been suggested for use in regenerating nervous systemtissue following spinal cord injury or trauma, such as brachial plexusinjury, neuroimmunologic disorders, such as acute or idiopathictransverse myelitis (TM), or any other disease or condition whereregeneration and/or protection of neurons or neural tissue is desired,since FGF-1 is believed to stimulate neural proliferation and growth andmay be neuroprotective. See, e.g., Lin P-S. et al., “Spinal CordImplantation with Acidic Fibroblast Growth Factor as a Treatment forRoot Avulsion in Obstetric Brachial Plexus Palsy,” J Chin Med Assoc68(8):392-396 (2005); Cheng, H. et al., “Spinal Cord Repair with AcidicFibroblast Growth Factor as a Treatment for a Patient with ChronicParaplegia,” SPINE 29(14):E284-E288 (2004); and Lin, P-H., “Functionalrecovery of chronic complete idiopathic transverse myelitis afteradministration of neurotrophic factors,” Spinal Cord 44:254-257 (2006),the entire contents and disclosures of which are hereby incorporated byreference.

Pharmaceutical or therapeutic administration of FGF proteins, such asFGF-1 and FGF-2, is limited by the fact that wild-type FGF proteins havepoor thermodynamic stability and a short half-life. See, e.g.,Szlachcic, A. et al., “Structure of a highly stable mutant of humanfibroblast growth factor 1,” Acta Cryst. D65:67-73 (2009), the entirecontents and disclosures of which are hereby incorporated by reference.For example, FGF-1 has poor thermodynamic stability with a meltingtemperature (i.e., a midpoint of thermal denaturation, or T_(m)) that isonly marginally above physiological temperature. See, e.g., Copeland, R.A., et al., “The structure of human acidic fibroblast growth factor andits interaction with heparin,” Archives of Biochemistry and Biophysics289:53-61 (1991), the entire contents and disclosure of which are herebyincorporated by reference. The functional half-life of wild-type FGF-1in unconditioned DMEM is only about 1.0 hour according to a culturedfibroblast proliferation assay. Although incubation experiments in TBSbuffer demonstrate aggregation and loss of soluble monomeric of FGF-1over a different time scale, these studies do show that loss of solublemonomeric FGF-1 protein over time is due to irreversible aggregationwith soluble FGF-1 protein showing formation of higher-mass disulfideadducts. Because of its intrinsic property of instability, FGF-1 isprone to both aggregation and proteolysis, which may causeimmunogenicity. Accordingly, substantial effort has been spent onidentifying appropriate formulations to counteract these intrinsicproperties often with mixed success.

FGF-1 is a “heparin-binding” growth factor, and upon binding of heparin,the T_(m) of FGF-1 increases by about 20° C., and heparin-bound FGF-1exhibits reduced susceptibility to denaturation-induced aggregation,thiol reactivity, and proteolytic degradation. See, e.g., Copeland R. A.et al. (1991), supra; and Gospodarowicz, D. et al., “Heparin protectsbasic and acidic FGF from inactivation,” Journal of Cellular Physiology128:475-484 (1986), the entire contents and disclosures of which arehereby incorporated by reference. Therefore, one approach to overcomingthe therapeutic limitation of FGF protein instability is to administerFGF-1 bound to heparin. Indeed, FGF-1 formulated with the addition ofheparin as a protein biopharmaceutical is currently in phase II clinicaltrials (NCT00117936) for proangiogenic therapy in coronary heartdisease. However, heparin adds considerable additional expense, has itsown pharmacological properties (e.g., it is an anti-coagulant), isderived from animal tissues (with associated concerns regarding thepotential for infectious agents), and causes adverse inflammatory orallergic reactions in a segment of the population. Thus, formulationefforts to modulate the physical properties of a protein are oftendifficult to achieve and can introduce undesired additional cost or sideeffects.

An alternative approach to “PEGylation” or formulation with heparin isto alter the physical properties of an FGF protein by mutagenesis. Bychanging its amino acid sequence, an FGF protein may have greaterthermodynamic stability and/or increased functional half-life as well asincreased solubility and resistance to proteolytic degradation,aggregation, or immunogenic potential. Mutating proteins to improvetheir properties for human therapeutic application is a viable approach.For example, over thirty mutant forms of proteins have been approved bythe FDA for use as human biopharmaceuticals. See, e.g., Kurtzman, A. L.et al., “Advances in directed protein evolution by recursive geneticrecombination: applications to therapeutic proteins” Curr Opin Biotech12:361-370 (2001), the entire contents and disclosures of which arehereby incorporated by reference. Examples include mutations thatcontribute to increased yields during purification, increased in vivofunctional half-life, or increased specific activity, such as mutationsof buried free-cysteine residues in β-interferon (Betaseron®) andinterleukin-2 (Proleukin®) as well as others hypothesized to increasethermostability. Thus, a mutational approach to improve the physicalproperties of proteins is a viable route to develop “second-generation”protein biopharmaceuticals having improved thermodynamic stabilityand/or functional half-life.

However, the concepts of thermodynamic stability and half-life areseparable properties of a protein. For example, as described furtherbelow, mutation of buried free cysteine residues of proteins mayincrease their functional half-life despite causing a reducedthermodynamic stability because mutation of these reactive free cysteineresidues avoids the irreversible formation of disulfide bonds that areincompatible with the native conformation of the protein. Therefore,combining separate mutations that eliminate free cysteines with othermutations that increase the thermostability of a protein may have asynergistic effect on the half-life of the protein by avoiding theirreversible denaturation pathway resulting from thiol reactivity of thefree cysteine (e.g., disulfide formation) while simultaneouslyincreasing the thermodynamic stability of the protein. This isespecially true considering that protein unfolding, which is dependenton protein stability, is often a necessary first step for theirreversible denaturation pathway resulting from exposure of thereactive free cysteine.

The fundamental FGF protein structure is described by a ˜120 amino aciddomain that forms a β-trefoil architecture. See, e.g., Murzin, A. G. etal., “β-Trefoil fold. Patterns of structure and sequence in the kunitzinhibitors interleukins-1β and 1α and fibroblast growth factors,”Journal of Molecular Biology 223:531-543 (1992), the entire contents anddisclosure of which are hereby incorporated by reference. Among the 22members of the mouse/human FGF family, three positions are absolutelyconserved, and include Gly71, Cys83, and Phe132 (using numbering schemeof the 140 amino acid form of FGF-1). Gly71 is located at the i+3position in a type 1 β-turn and is the statistically-preferred residueat this position due to structural considerations of backbone strain.See, e.g., Hutchinson, E. G. et al., “A revised set of potentials forbeta-turn formation in proteins,” Protein Sci 3:2207-16 (1994);Guruprasad, K. et al., “Beta- and gamma-turns in proteins revisited: anew set of amino acid turn-type dependent positional preferences andpotentials,” J Biosci 25:143-56 (2000); Kim, J. et al., “Identificationof a key structural element for protein folding within β-hairpin turns,”Journal of Molecular Biology 328:951-961 (2003); and Lee, J. et al., “Alogical OR redundancy with the Asx-Pro-Asx-Gly type 1 β-turn motif,”Journal of Molecular Biology 377:1251-1264 (2008), the entire contentsand disclosures of which are hereby incorporated by reference.

Phe132 is a large aromatic residue that forms part of the hydrophobiccore of the protein. Such large hydrophobic residues within the proteininterior make a substantial contribution to protein stability. See,e.g., Shortle, D. et al., “Contributions of the Large Hydrophobic AminoAcids to the Stability of Staphylococcal Nuclease,” Biochemistry29:8033-8041 (1990); Eriksson, A. E. et al., “Response of a proteinstructure to cavity-creating mutations and its relation to thehydrophobic effect,” Science 255:178-183 (1992); and Brych, S. R. etal., “Structure and stability effects of mutations designed to increasethe primary sequence symmetry within the core region of a β-trefoil,”Protein Science 10:2587-2599 (2001), the entire contents and disclosuresof which are hereby incorporated by reference. Cys83 is located at asolvent-inaccessible position in the protein and therefore has noidentifiable role related to receptor-binding functionality, neitherdoes it provide significant buried hydrophobic area that mightcontribute to stability.

FGF-1 contains three free-cysteine residues at positions 16, 83, and 117(using the 140 amino acid numbering scheme of human FGF-1) that maylimit the functional stability due to reactive thiol chemistry. See,e.g., Ortega, S. et al., “Conversion of cysteine to serine residuesalters the activity, stability, and heparin dependence of acidicfibroblast growth factor,” Journal of Biological Chemistry 266:5842-5846(1991); and Estape, D., et al., “Susceptibility towards intramoleculardisulphide-bond formation affects conformational stability and foldingof human basic fibroblast growth factor,” Biochem J 335:343-9 (1998),the entire contents and disclosures of which are hereby incorporated byreference.

Cysteine is the second-least abundant amino acid in proteins (aftertryptophan), yet is among the most highly conserved in functionallyimportant sites involving catalysis, regulation, cofactor binding, andstability. See, e.g., Fomenko, D. E. et al., “Functional diversity ofcysteine residues in proteins and unique features of catalyticredox-active cysteines in thiol oxidoreductases,” Mol Cells 26:228-35(2008), the entire contents and disclosure of which are herebyincorporated by reference. The unique properties of cysteine have theirbasis in the side chain Sγ sulfur atom participating in a variety ofdifferent functional roles, including disulfide bond formation,metal-binding, electron donation, hydrolysis, and redox-catalysis.However, some cysteine residues do not participate in these functionalroles and exist instead as structural free-cysteines within the protein.These free cysteines are approximately evenly distributed betweeninterior and solvent exposed positions of proteins. See, e.g., Petersen,M. T. N. et al., “Amino acid neighbours and detailed conformationalanalysis of cysteines in proteins,” Protein Engineering 12:535-548(1999), the entire contents and disclosure of which are herebyincorporated by reference.

Published work has made note of the fact that free-cysteine residueswithin the interior of a protein may effectively limit the protein'sfunctional half-life. Free cysteines have chemically reactive thiolsthat are subject to chemical modification (e.g., covalent disulfide bondformation) should they become exposed (i.e., solvent-accessible orpresent on the protein surface), which occurs transiently during thedynamic equilibrium process of maintaining protein structure. Thechemical reactivity of these free cysteine residues may present majorstructural difficulties for accommodation within the native proteininterior and may result in an irreversible unfolding pathway when thesefree cysteine residues become exposed. Free-cysteine residues arechemically reactive thiols that are subject to covalent bond formationwith other reactive thiols. If present on the solvent-accessible surfaceof a protein, a free cysteine may potentially participate in a disulfideadduct while the protein maintains its native conformation. However,when present within the solvent-inaccessible core, substantialstructural rearrangement generally must occur to permit accessibilityand reactivity. Conversely, the formation of a disulfide adductinvolving a buried cysteine is typically structurally incompatible withthe native conformation and results in misfolded forms of the proteinthat may promote aggregation and increased immunogenicity.

An analysis of a set of 131 non-homologous single-domain protein X-raystructures (1.95 Å resolution or better) has shown that the prevalenceof free-cysteine residues in proteins is about 0.5% (i.e., about onefree-cysteine in an average size protein). Furthermore, 50% of thesefree-cysteines are buried within the protein interior. See, e.g.,Petersen, M. T. N. et al., “Amino acid neighbours and detailedconformational analysis of cysteines in proteins,” Protein Engineering12:535-548 (1999), the entire contents and disclosure of which arehereby incorporated by reference. Thus, although potentiallyhighly-problematic for protein therapeutic application, the presence ofburied free-cysteines in proteins is a surprisingly common occurrence.Examples of potentially therapeutic proteins having buried freecysteines include fibroblast growth factors, interleukin-2,β-interferon, granulocyte colony stimulating factor, and insulin-likegrowth factor-binding protein-1 (with the majority of these beingapproved human therapeutics).

Due to the potential negative consequences to protein structure causedby thiol adduct formation of the buried free cysteines at positions 16and/or 83 and/or the partially accessible free cysteine at position 117of human FGF-1 (based on the 140 amino acid numbering scheme), mutationto eliminate one or more of these free cysteine residues has been shownto produce a notable increase in the functional half-life of FGF-1protein. See, e.g., Culajay, J. F. et al., “Thermodynamiccharacterization of mutants of human fibroblast growth factor 1 with anincreased physiological half-life,” Biochemistry 39:7153-7158 (2000);Ortega, S. et al. (1991), supra; Lee, J. et al., “The interactionbetween thermostability and buried free cysteines in regulating thefunctional half-life of fibroblast growth factor-1,” J. Mol. Biol.393:113-127 (2009); Cuevas, P. et al., “Hypotensive activity offibroblast growth factor.” Science 254:1208-10 (1991); and U.S. Pat. No.5,409,897 (Thomas et al.; issued Apr. 25, 1995), the entire contents anddisclosures of which are hereby incorporated by reference.

The 22 members of the mouse/human FGF family of proteins contain aconserved cysteine residue at position 83 (using the 140 amino acidnumbering scheme for human FGF-1). Sequence and available structuralinformation suggests that this position is a free cysteine in 16 membersincluding the FGF-1 family, such as FGF-1 and FGF-2, but participates asa half-cystine (i.e., in a disulfide bond) in at least 3 members (andpossibly as many as 6). For example, position 66 (using the same 140amino acid numbering scheme) is a cysteine residue at correspondingpositions of FGF-8, 19 and 23 and has been shown to form a half-cystinewith the cysteine at a position corresponding to position 83 of FGF-1.

According to embodiments of the present invention, a previouslyunreported mutation of alanine (Ala) at position 66 of human FGF-1(using the 140 amino acid numbering scheme for human FGF-1) or acorresponding position of a protein encoded by a FGF-1 subfamily memberto cysteine (Cys) is provided to encourage formation of a disulfide bondwith the cysteine at a position corresponding to position 83 of humanFGF-1. In contrast to previous reports of stabilization of FGF proteinsachieved with removal of buried free cysteine residues, replacement ofthe alanine at position 66 of FGF-1 with a cysteine by mutation is shown(in greater detail below) to increase the stability of FGF-1 proteinunder oxidizing conditions despite some strain in protein structure toaccommodate the mutation. Based on these results, the absence of acysteine residue at position 66 of some wild-type FGF proteins mayensure that the cysteine residue at position 83 remains a free cysteinethat functions as a buried reactive thiol to regulate (i.e., reduce) thefunctional half-life of the protein. Limiting the functional half-lifeof FGF proteins by maintaining a free cysteine at position 83 may beimportant for some FGF proteins considering that FGF proteins aregenerally potent mitogens. Thus, mutation of alanine at position 66 ofhuman FGF-1 (or mutation at a position of other FGF proteinscorresponding to position 66 of human FGF-1) to cysteine is providedherein as a way to increase the functional half-life of FGF proteinsgenerally for improved pharmaceutical or therapeutic application.

Despite its similarity to other FGF proteins, it is somewhat surprisingthat mutation of alanine at position 66 of human FGF-1 to cysteine wouldresult in a net stabilization of the protein. Replacement of alanine atposition 66 with cysteine is divergent from the wild-type human FGF-1,and random mutations are generally more likely to destabilize ratherthan stabilize protein structure. In fact, the Ala66→Cys mutation isitself destabilizing by about 5.1 kJ/mol under reducing conditions whenthe cysteine is a free cysteine, but the mutation appears to onlyprovide net stabilization under oxidizing conditions (by about 13.6kJ/mol) upon formation of a disulfide bond. Given that there are otherstructural and amino acid differences among FGF members, it cannot beassumed that a net stabilizing disulfide bond would necessarily formwith mutation of Ala at position 66 of FGF-1. Thus, it is not expectednecessarily that the Ala66→Cys mutation would result in two cysteineresidues that are ideally juxtaposed to form a net stabilizing disulfidebond. In fact, a software program (Disulfide by Design, Version 1.2)designed to predict whether the creation of a cysteine residue atspecific positions within a protein might create novel disulfide bondsdid not identify positions 66 and 83 of human FGF-1 as candidates.

Furthermore, mutation of alanine at position 66 of human FGF-1 tocysteine is in contrast to the teachings of U.S. Pat. No. 5,409,897(Thomas et al.; issued Apr. 25, 1995). The '897 Thomas patent describesthe removal of free cysteines of FGF-1 by mutation at positions 16, 83,and 117 to eliminate their thiol reactivity, whereas embodiments of thepresent invention describe mutation of alanine at position 66 of humanFGF-1 to create a cysteine residue at this position, which maycontribute to an increased functional half-life of a mutant FGF proteinby forming a disulfide bond with a cysteine partner. Thus, mutation ofalanine at position 66 of human FGF-1 to cysteine requires the retentionof a cysteine residue at position 83 to allow for disulfide bondformation in further contrast with the '897 Thomas patent. Suchformation of a disulfide bond may increase the thermostability of theprotein while simultaneously eliminating thiol reactivity of the freecysteine at position 83.

According to embodiments of the present invention, mutations may beintroduced into a FGF protein, such as FGF-1 or FGF-2, to sufficientlyincrease the thermodynamic stability of the protein to avoid any needfor the use of heparin. According to embodiments of the presentinvention, mutations may be introduced into the interior or core of aFGF protein, such as FGF-1 or FGF-2, to increase thermodynamicstability, such that structural changes may be accommodated within theprotein interior without any significant structural change to thesurface of the protein that might otherwise trigger immunogenicity.Greater thermostability of FGF proteins may be achieved by introducingmutations within the solvent-excluded interior of the protein thateliminate or improve upon packing defects within the wild-typestructure. Significant stability gains may be realized using thisstrategy, and these increases in thermostability may be achieved withminimal perturbation of the overall wild-type FGF protein structure,including surface features and solvent structure. Such mutations providea protein design strategy whereby functional half-life may bemanipulated while minimizing immunogenic potential or need for the useof heparin.

Human FGF-1 has a β-trefoil tertiary structure with a pseudo-threefoldβ-barrel axis of symmetry composed of antiparallel β strands around ahydrophobic core. Mutations have been made to core packing residues ofhuman FGF-1 protein to study the effects of increased symmetry betweencore packing residues of the three structural units. Several of thesecore packing mutations may increase the thermodynamic stability of humanFGF-1 protein, such as by reducing microcavities within the core of theprotein. For example, the following mutations have been made to the coreregion of human FGF-1 with varying effects on stability (positions referto those within human FGF-1 using the 140 amino acid numbering scheme):Leu44→Phe; Met67→Ile; Leu73→Val; Val109→Leu; Leu111→Ile; Cys117→Val; andcombinations thereof. Some examples of mutant combinations that havealso have been made include: (Sym 2) Leu73→Val+Val109→Leu; (Sym 3)Leu44→Phe+Leu73→Val+Val109→Leu; (Sym 4)Leu44→Phe+Leu73→Val+Val109→Leu+Cys117→Val; (Sym 5)Leu44→Phe+Leu73→Val+Val109→Leu+Leu111→Ile+Cys117→Val; and (Sym 6)Leu44→Phe+Met67→Ile+Leu73→Val+Val109→Leu+Leu111→Ile+Cys117→Val. See,e.g., Brych S. R., et al., “Structure and stability effects of mutationsdesigned to increase the primary sequence symmetry within the coreregion of a β-trefoil,” Protein Science 10:2587-2599 (2001); and BrychS. R., et al., “Accommodation of a highly symmetric protein superfold,”Protein Science 12:2704-2718 (2003), the entire contents and disclosuresof which are hereby incorporated by reference.

However, the Met at position 67 appears highly intolerant tosubstitution in the Sym6 mutant and exhibited precipitation duringpurification. Separately, it is believed that the instability withmutation at position 67 is the result of two apparent insertions ofadjacent loops involving amino acids 104-106 and 120-122 that distortthe tertiary structure of FGF-1. Therefore, deleting these insertionsmight impart greater stability. Indeed, combining the Sym6 mutant abovewith deletions of amino acid positions 104-106 and 120-122 along withAla103→Gly and Arg119→Gly mutations (Sym6ΔΔ) resulted in a FGF-1 proteinhaving greater stability and mitogenic activity than even wild-typeFGF-1. See, e.g., Brych, S. R. et al., “Symmetric Primary and TertiaryStructure Mutations within a Symmetric Superfold: A Solution, not aConstraint, to Achieve a Foldable Polypeptide,” J. Mol. Biol.344:769-780 (2004), the entire contents and disclosure of which arehereby incorporated by reference.

Other mutational approaches have been taken to increase the stability ofFGF-1 protein. In addition to the core packing residues, there aresymmetrically related pairs of buried hydrophobic residues in FGFprotein (termed “mini-cores”) that are not part of the central core. Inhuman FGF-1, these include symmetry related positions 22, 64, and 108and symmetry related positions 42, 83, and 130 (positions refer to thosewithin human FGF-1 using the 140 amino acid numbering scheme). Forexample, the following mutations have been made to the mini-core regionsof human FGF-1 with varying effects on stability (positions refer tothose within human FGF-1 using the 140 amino acid numbering scheme):Ile42→Cys; Cys83→Ile; Ile130→Cys; Phe22→Tyr; Tyr64→Phe; and Phe108→Tyr,and combinations thereof. See, e.g., Dubey, V. K., et al., “Redesigningsymmetry-related “mini-core” regions of FGF-1 to increase primarystructure symmetry: Thermodynamic and functional consequences ofstructural symmetry,” Protein Science 14:2315-2323 (2005), the entirecontents and disclosure of which are hereby incorporated by reference.

The N-terminus and C-terminus of human FGF-1 are composed of β strandsthat hydrogen bond to one another between residues 13 through 17 andresidues 131 through 135 (positions refer to those within human FGF-1using the 140 amino acid numbering scheme). However, this region ofhuman FGF-1 has two solvent-excluded microcavities that may destabilizethe protein. To study the effects on stability, mutations have been madeto lysine at position 12 and proline at position 134 of human FGF-1. Forexample, the following mutations have been made to human FGF-1 (usingthe 140 amino acid numbering scheme for human FGF-1): Lys12→Cys;Lys12→Thr; Lys12→Val; Pro134→Cys; Pro134→Thr; and Pro134→Val. Inaddition, the Lys12→Val and Pro134→Val mutations may be combined withsymmetry related mutations (based on the three-fold pseudo-symmetryarchitecture of FGF-1) Asn95→Val (for Lys12→Val) and Leu46→Val andGlu87→Val (for Pro134→Val), and combinations thereof. These mutationsgenerally increase the thermostability and mitogenicity of FGF-1 tovarying degrees. See, e.g., Dubey, V. K. et al., “Spackling the Crack:Stabilizing Human Fibroblast Growth Factor-1 by Targeting the N and Cterminus β-Strand Interactions,” J. Mol. Biol. 371:256-268 (2007), theentire contents and disclosure of which are hereby incorporated byreference.

According to embodiments of the present invention, a previouslyunreported mutation of the core packing phenylalanine (Phe) at position132 of human FGF-1 (using the 140 amino acid numbering scheme for humanFGF-1) or a corresponding position of a protein encoded by a FGF-1subfamily member to tryptophan (Trp) is provided. Replacement of thephenylalanine (Phe) at position 132 of human FGF-1 with tryptophan (Trp)by mutation is shown (in greater detail below) to increase the stabilityof the FGF-1 protein.

Although the phenylalanine (Phe) at position 132 of human FGF-1 is acore packing residue which may be amenable to mutation, not allmutations that replace core packing residues of FGF-1 with otherhydrophobic amino acids improve thermodynamic stability of FGF-1. Thereare many uncertainties that make prediction of stabilizing mutationswithin the core of FGF-1 difficult. With regard to the Phe132→Trpmutant, while both phenylalanine and tryptophan are large aromatic sidechains that may be good choices to bury within the core of FGF-1,tryptophan is different from phenylalanine in that it has ahydrogen-bonding requirement with the hydrogen bound to the nitrogen ofthe five member indole ring acting as a hydrogen bond donor. On thesurface of a protein, solvent molecules may act as hydrogen bondacceptors to partner with the NH donor of the indole ring. However,within the interior of the protein (as with Phe 132 of FGF-1), otherstructural portions of the protein must serve as the hydrogen bondingpartner. Therefore, it cannot be assumed that there would be a hydrogenbonding acceptor that is already positioned and ideally juxtaposed toPhe 132 of FGF-1, such that the Phe132→Trp mutation may be accommodatedwith minimal structural strain. As it turns out, the lone pair ofelectrons on the oxygen of the main chain carbonyl group at position Val109 is positioned to act as a hydrogen bond acceptor for the newlyintroduced Trp at position 132 of FGF-1.

Furthermore, the Trp residue introduced by mutation is larger than thePhe at position 132 of wild-type FGF-1, and thus it cannot be assumedthat the protein environment would necessarily be able to accommodatethe larger Trp while satisfying the additional hydrogen bondingrequirements. For example, a symmetry-related Phe85→Trp mutation isaccommodated with an actual loss of favorable van der Waalsinteractions. Thus, disruption of local van der Waals interactions toaccommodate a larger aromatic mutant side chain may offset any gain fromthe additional buried area within the protein interior. In addition,Phe132 is one of only three residues that are known to be absolutelyconserved among the 22 mouse/human FGF family members, which may suggestthat FGF proteins would be intolerant to mutation at the Phe132position, and large hydrophobic residues within the protein interiorgenerally make a substantial contribution to protein stability.

However, according to X-ray structural data (see below), the Phe132→Trpmutation side chain partially fills two cavities located adjacent topositions 132 with minimal perturbation of the surrounding structure.Accordingly, the Phe132→Trp mutation is found to increasethermostability of FGF-1 presumably due to greater space filling withinthe core and accommodation of the hydrogen bonding requirements of Trpalong with minimal perturbation of the protein structure. Thus, mutationof the phenylalanine at position 132 of human FGF-1 (or mutation at thecorresponding position of other FGF proteins) to tryptophan is providedherein as a way to increase the stability and/or functional half-life ofFGF proteins generally for improved pharmaceutical or therapeuticapplication.

Wild-type human FGF-1 may exist in multiple forms of varying length invivo due to proteolytic processing. Human Fgf-1 mRNA encodes a 155 aminoacid protein (SEQ ID NO: 2; see also FIG. 1). However, a 154 amino acidform of human FGF-1 (SEQ ID NO: 3) may be formed from the 155 amino acidform if the starting methionine is removed. (See underlined methioninein FIG. 1) In addition, the 140 amino acid form of wild-type human FGF-1(SEQ ID NO: 1; see also FIG. 2) is formed in vivo by proteolyticprocessing of the longer forms of FGF-1 to remove the first 15 or 14amino acids of the 155 amino acid or the 154 amino acid forms,respectively (see shaded sequence of FIG. 1).

According to a broad aspect of the present invention, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 90% identical to the polypeptide sequence of amammalian FGF-1 subfamily protein, or a functional fragment thereof,wherein the alanine (Ala) at an amino acid position of the mutant FGFprotein corresponding to amino acid position 66 of wild-type human FGF-1(based on the 140 amino acid numbering scheme of human FGF-1) isreplaced with cysteine (Cys).

According to embodiments of the present invention, a mutant fibroblastgrowth factor (FGF) protein is provided having a polypeptide sequencethat is at least 90% identical to the 140 amino acid polypeptidesequence of wild-type human FGF-1 protein (SEQ ID NO: 1), or afunctional fragment thereof, wherein the alanine (Ala) at an amino acidposition of the mutant FGF protein corresponding to amino acid position66 of wild-type human FGF-1 (based on the 140 amino acid numberingscheme of human FGF-1) is replaced with cysteine (Cys). Alternatively, amutant fibroblast growth factor (FGF) protein is provided having apolypeptide sequence that is at least 95% identical to the 140 aminoacid polypeptide sequence of wild-type human FGF-1 protein (SEQ ID NO:1), or a functional fragment thereof, wherein the alanine (Ala) at anamino acid position of the mutant FGF protein corresponding to aminoacid position 66 of wild-type human FGF-1 (based on the 140 amino acidnumbering scheme of human FGF-1) is replaced with cysteine (Cys). Forexample, according to some embodiments, a mutant FGF protein may be SEQID NO: 5, or a functional fragment thereof.

According to some embodiments, a mutant fibroblast growth factor (FGF)protein is provided having a polypeptide sequence that is at least 90%identical to the 155 amino acid polypeptide sequence of wild-type humanFGF-1 protein (SEQ ID NO: 2), or a functional fragment thereof, whereinthe alanine (Ala) at a position of the mutant FGF protein correspondingto position 81 of wild-type human FGF-1 (based on the 155 amino acidnumbering scheme of human FGF-1) is replaced with cysteine (Cys).According to some embodiments, the starting methionine of the 155 aminoacid form of wild-type human FGF-1 may be absent in the mutant FGFprotein. According to these embodiments, a mutant fibroblast growthfactor (FGF) protein is provided having a polypeptide sequence that isat least 90% identical to the 154 amino acid polypeptide sequence ofwild-type human FGF-1 protein (SEQ ID NO: 3), or a functional fragmentthereof, wherein the alanine (Ala) at a position of the mutant FGFprotein corresponding to position 80 of wild-type human FGF-1 (based onthe 154 amino acid numbering scheme of human FGF-1) is replaced withcysteine (Cys).

According to some embodiments of the present invention, the Ala to Cysmutation or substitution at a position of the mutant FGF proteincorresponding to position 66 of human FGF-1 (based on the 140 amino acidnumbering scheme of human FGF-1) may be combined with one or more otherknown mutation(s), substitution(s), modification(s), deletion(s), etc.According to some embodiments, the Ala to Cys mutation or substitutionat position 66 (based on the 140 amino acid numbering scheme of humanFGF-1) may be combined with one or more mutation(s) or substitution(s)to replace one or both of the free Cys residues at positions 16 and 117of wild-type human FGF-1 with a different amino acid, such as alanine(Ala), serine (Ser), threonine (Thr), valine (Val), or isoleucine (Ile),in the mutant FGF protein. Presumably, mutation of the Cys residue atposition 83 would remove the benefit of the Ala66→Cysmutation/substitution in the mutant FGF-1 by eliminating its proposedcystine partner. According to similar alternative embodiments, a mutantFGF protein may include the same combinations of mutations at positionscorresponding to positions Cys30, Ala80, and Cys131 (based on the 154amino acid form of human FGF-1) or Cys31, Ala81, and Cys132 (based onthe 155 amino acid form of human FGF-1). One skilled in the art would beable to determine the polypeptide sequences for mutant FGF proteinshaving each of these mutant combinations based on the disclosureprovided herein.

According to some embodiments, the Ala to Cys mutation or substitutionat a position of a mutant FGF protein corresponding to position 66 ofhuman FGF-1 (based on the 140 amino acid numbering scheme of humanFGF-1) may be combined with one or more mutation(s) or substitution(s)to replace core packing residue(s) to increase the thermodynamicstability of the mutant FGF protein. For example, the Ala to Cysmutation or substitution at position 66 (based on the 140 amino acidnumbering scheme of human FGF-1) may be combined with one or more of thefollowing mutations or substitutions of core packing residue(s):Leu44→Phe; Met67→Ile; Leu73→Val; Val109→Leu; Leu111→Ile; and/orCys117→Val. For example, the Ala to Cys mutation or substitution atposition 66 (based on the 140 amino acid numbering scheme of humanFGF-1) may be combined with one or more of the mutations orsubstitutions of core packing residues, which may be combined with otherstabilizing mutations, such as the following: Leu44→Phe+Ala66→Cys;Ala66→Cys+Leu73→Val+Val109→Leu;Leu44→Phe+Ala66→Cys+Leu73→Val+Val109→Leu;Leu44→Phe+Ala66→Cys+Leu73→Val+Val109→Leu+Cys117→Val;Leu44→Phe+Ala66→Cys+Leu73→Val+Val109→Leu+Leu111→Ile+Cys117→Val;Leu44→Phe+Ala66→Cys+Met67→Ile+Leu73→Val+Val109→Leu+Leu111→Ile+Cys117→Val;or Leu44→Phe+Ala66→Cys+Met67→Ile+Leu73→Val+Ala103→Gly+104-106deletion+Val109→Leu+Leu111→Ile+Cys117→Val+Arg119→Gly+120-122 deletion.The same mutant combinations may be applied at corresponding positionsof mutant FGF proteins having high percent identity with the 154 and 155amino acid forms of human FGF-1. One skilled in the art would be able todetermine the polypeptide sequences of these mutant FGF proteins basedon the disclosure provided herein.

According to some embodiments, the Ala to Cys mutation or substitutionat a position corresponding to position 66 of FGF-1 (based on the 140amino acid numbering scheme of human FGF-1) may be combined with one ormore mutation(s) or substitution(s) to replace “mini-core” residue(s) toincrease the thermodynamic stability of the mutant FGF protein. The Alato Cys mutation or substitution at position 66 (based on the 140 aminoacid numbering scheme of human FGF-1) may be combined with one or moreof the following mutation(s) or substitution(s) of “mini-core”residue(s): Ile42→Cys; Cys83→Ile; Ile130→Cys; Phe22→Tyr; Tyr64→Phe;and/or Phe108→Tyr. For example, the Ala to Cys mutation or substitutionat position 66 (based on the 140 amino acid numbering scheme of humanFGF-1) may be combined with one or more of the mutation(s) orsubstitution(s) of “mini-core” residue(s), such as the following:Phe22→Tyr+Ala66→Cys+Phe108→Tyr; Ile42→Cys+Ala66→Cys+Ile130→Cys; orPhe22→Tyr+Ile42→Cys+Ala66→Cys+Phe108→Tyr+Ile130→Cys.

In addition, the Ala to Cys mutation or substitution at position 66(based on the 140 amino acid numbering scheme of human FGF-1) may becombined with one or more of the mutation(s) or substitution(s) of“mini-core” residue(s) in further combination with core packingmutations, such as the following:Phe22→Tyr+Leu44→Phe+Ala66→Cys+Met67→Ile+Leu73→Val+Ala103→Gly+104-106deletion+Phe108→Tyr+Val109→Leu+Leu111→Ile+Cys117→Val+Arg119→Gly+120-122deletion;Ile42→Cys+Leu44→Phe+Ala66→Cys+Met67→Ile+Leu73→Val+Ala103→Gly+104-106deletion+Val109→Leu+Leu111→Ile+Cys117→Val+Arg119→Gly+120-122deletion+Ile130→Cys; orPhe22→Tyr+Ile42→Cys+Leu44→Phe+Ala66→Cys+Met67→Ile+Leu73→Val+Ala103→Gly+104-106deletion+Phe108→Tyr+Val109→Leu+Leu111→Ile+Cys117→Val+Arg119→Gly+120-122deletion+Ile130→Cys. The same mutant combinations may be made atcorresponding positions of mutant FGF proteins having a similarly highpercent identity (e.g., at least 90% identity) with the 154 and 155amino acid forms of human FGF-1. One skilled in the art would be able todetermine the polypeptide sequences of these mutant FGF proteins basedon the disclosure provided herein.

According to some embodiments, the Ala to Cys mutation or substitutionat a position corresponding to position 66 of human FGF-1 (based on the140 amino acid numbering scheme of human FGF-1) may be combined with oneor more mutation(s) or substitution(s) to replace one or more ofN-terminal residues 13 through 17 and/or C-terminal residues 131 through135 (using the 140 amino acid numbering scheme for human FGF-1) toincrease the thermostability of the mutant FGF protein. The Ala to Cysmutation or substitution at position 66 (based on the 140 amino acidnumbering scheme of human FGF-1) may be combined with one or more of thefollowing mutation(s) or substitution(s) of these N-terminal and/orC-terminal residues: Lys12→Cys; Lys12→Thr; Lys12→Val; Leu46→Val;Glu87→Val; Asn95→Val; Pro134→Cys; Pro134→Thr; and/or Pro134→Val. Forexample, the Ala to Cys mutation or substitution at position 66 (basedon the 140 amino acid numbering scheme of human FGF-1) may be combinedwith one or more of the mutation(s) or substitution(s) of N-terminaland/or C-terminal residue(s), which may be combined with mutations atsymmetry related positions, such as the following:Lys12→Val+Ala66→Cys+Pro134→Val; Lys12→Val+Ala66→Cys+Asn95→Val;Leu46→Val+Ala66→Cys+Pro134→Val; Ala66→Cys+Glu87→Val+Pro134→Val;Leu46→Val+Ala66→Cys+Glu87→Val+Pro134→Val; orLys12→Val+Leu46→Val+Ala66→Cys+Glu87→Val+Asn95→Val+Pro134→Val. The samemutant combinations may be applied at corresponding positions of mutantFGF proteins having a high percent identity (e.g., at least 90%identity) with the 154 and 155 amino acid forms of human FGF-1. Oneskilled in the art would be able to determine the polypeptide sequencesof these mutant FGF proteins based on the disclosure provided herein.

According to some embodiments of the present invention, the Ala to Cysmutation or substitution at a position of the mutant FGF proteincorresponding to position 66 of human FGF-1 (based on the 140 amino acidnumbering scheme of human FGF-1) may be combined with two or moremutations or substitutions of different types. For example, the Ala toCys mutation or substitution at a position of the mutant FGF proteincorresponding to position 66 of human FGF-1 (based on the 140 amino acidnumbering scheme of human FGF-1) may be combined with any combination ofmutation or substitution of the following residue types: free cysteineresidues, core packing residues, “mini-core” residues, and/orinteracting N-terminal or C-terminal residues. Indeed, the Ala to Cysmutation or substitution at a position of the mutant FGF proteincorresponding to position 66 of human FGF-1 (based on the 140 amino acidnumbering scheme of human FGF-1) may be combined with any combination ofmutation(s), modification(s), substitution(s), deletion(s), etc., knownin the art or described herein.

According to another broad aspect of the present invention, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 90% identical to the polypeptide sequence of amammalian FGF-1 subfamily protein, or a functional fragment thereof,wherein the phenylalanine (Phe) at an amino acid position of the mutantFGF protein corresponding to amino acid position 132 of wild-type humanFGF-1 (based on the 140 amino acid numbering scheme of human FGF-1) isreplaced with tryptophan (Trp).

According to some embodiments of the present invention, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 90% identical to the polypeptide sequence ofthe 140 amino acid polypeptide sequence of wild-type human FGF-1 protein(SEQ ID NO: 1) or a functional fragment thereof, wherein thephenylalanine (Phe) at an amino acid position of the mutant FGF proteincorresponding to amino acid position 132 of wild-type human FGF-1 (basedon the 140 amino acid numbering scheme of human FGF-1) is replaced withtryptophan (Trp). Alternatively, a mutant fibroblast growth factor (FGF)protein is provided having a polypeptide sequence that is at least 95%identical to the 140 amino acid polypeptide sequence of wild-type humanFGF-1 protein (SEQ ID NO: 1) or a functional fragment thereof, whereinthe phenylalanine (Phe) at an amino acid position of the mutant FGFprotein corresponding to amino acid position 132 of wild-type humanFGF-1 (based on the 140 amino acid numbering scheme of human FGF-1) isreplaced with tryptophan (Trp). For example, according to someembodiments, a mutant FGF protein may be SEQ ID NO: 7, or a functionalfragment thereof.

According to some embodiments, a mutant fibroblast growth factor (FGF)protein is provided having a polypeptide sequence that is at least 90%identical to the 155 amino acid polypeptide sequence of wild-type humanFGF-1 protein (SEQ ID NO: 2) or a functional fragment thereof, whereinthe phenylalanine (Phe) at an amino acid position of the mutant FGFprotein corresponding to amino acid position 147 of wild-type humanFGF-1 (based on the 155 amino acid numbering scheme of human FGF-1) isreplaced with tryptophan (Trp). According to some embodiments, thestarting methionine of the 155 amino acid form of wild-type human FGF-1may be absent from the mutant FGF protein. According to theseembodiments, a mutant fibroblast growth factor (FGF) protein is providedhaving a polypeptide sequence that is at least 90% identical to the 154amino acid polypeptide sequence of wild-type human FGF-1 protein (SEQ IDNO: 3) or a functional fragment thereof, wherein the phenylalanine (Phe)at an amino acid position of the mutant FGF protein corresponding toamino acid position 146 of wild-type human FGF-1 (based on the 154 aminoacid numbering scheme of human FGF-1) is replaced with tryptophan (Trp).

According to some embodiments of the present invention, the Phe to Trpmutation or substitution at a position corresponding to position 132 ofhuman FGF-1 (based on the 140 amino acid numbering scheme of humanFGF-1) of the mutant FGF protein may be combined with one or more otherknown mutation(s), substitution(s), modification(s), deletion(s), etc.According to some embodiments, the Phe to Trp mutation or substitutionat position 132 (based on the 140 amino acid numbering scheme of humanFGF-1) may be combined with one or more mutation(s) or substitution(s)to replace one or both of the free Cys residues at positions 16, 83, and117 of wild-type human FGF-1 with a different amino acid, such asalanine (Ala), serine (Ser), threonine (Thr), valine (Val), orisoleucine (Ile), in the mutant FGF protein. According to similarembodiments, a mutant FGF protein may include the same combinations ofmutations at positions corresponding to positions Cys30, Cys97, Cys131,and Phe146 (based on the 154 amino acid form of human FGF-1) or Cys31,Cys98, Cys132, and Phe147 (based on the 155 amino acid form of humanFGF-1). One skilled in the art would be able to determine thepolypeptide sequences for mutant FGF proteins having each of thesemutant combinations based on the disclosure provided herein.

According to some embodiments, the Phe to Trp mutation or substitutionat a position corresponding to position 132 of human FGF-1 (based on the140 amino acid numbering scheme of human FGF-1) may be combined with oneor more mutation(s) or substitution(s) to replace core packingresidue(s) to increase the thermodynamic stability of the mutant FGFprotein. For example, the Phe to Trp mutation or substitution atposition 132 (based on the 140 amino acid numbering scheme of humanFGF-1) may be combined with one or more of the following mutations- orsubstitutions of core packing residue(s): Leu44→Phe; Met67→Ile;Leu73→Val; Val109→Leu; Leu111→Ile; and/or Cys117→Val. For example, thePhe to Trp mutation or substitution at position 132 (based on the 140amino acid numbering scheme of human FGF-1) may be combined with one ormore of the mutations or substitutions of core packing residues, whichmay be combined with other stabilizing mutations, such as the following:Leu44→Phe+Phe132→Trp; Leu73→Val+Val109→Leu+Phe132→Trp;Leu44→Phe+Leu73→Val+Val109→Leu+Phe132→Trp;Leu44→Phe+Leu73→Val+Val109→Leu+Cys117→Val+Phe132→Trp;Leu44→Phe+Leu73→Val+Val109→Leu+Leu111→Ile+Cys117→Val+Phe132→Trp;Leu44→Phe+Met67→Ile+Leu73→Val+Val109→Leu+Leu111→Ile+Cys117→Val+Phe132→Trp;or Leu44→Phe+Met67→Ile+Leu73→Val+Ala103→Gly+104-106deletion+Val109→Leu+Leu111→Ile+Cys117→Val+Arg119→Gly+120-122deletion+Phe132→Trp. The same mutant combinations may be made atcorresponding positions of mutant FGF proteins having high percentidentity with the 154 and 155 amino acid forms of human FGF-1. Oneskilled in the art would be able to determine the polypeptide sequencesof these mutant FGF proteins based on the disclosure provided herein.

According to some embodiments, the Phe to Trp mutation or substitutionat a position corresponding to position 132 of human FGF-1 (based on the140 amino acid numbering scheme of human FGF-1) may be combined with oneor more mutation(s) or substitution(s) to replace “mini-core” residue(s)to increase the thermodynamic stability of the mutant FGF protein. Forexample, the Phe to Trp mutation or substitution at position 132 (basedon the 140 amino acid numbering scheme of human FGF-1) may be combinedwith one or more of the following mutation(s) or substitution(s) of“mini-core” residue(s): Ile42→Cys; Cys83→Ile; Ile130→Cys; Phe22→Tyr;Tyr64→Phe; and/or Phe108→Tyr. For example, the Phe to Trp mutation orsubstitution at position 132 (based on the 140 amino acid numberingscheme of human FGF-1) may be combined with one or more of themutation(s) or substitution(s) of “mini-core” residue(s), such as thefollowing: Phe22→Tyr+Phe108→Tyr+Phe132→Trp;Ile42→Cys+Ile130→Cys+Phe132→Trp; orPhe22→Tyr+Ile42→Cys+Phe108→Tyr+Ile130→Cys+Phe132→Trp. In addition, thePhe to Trp mutation or substitution at position 132 (based on the 140amino acid numbering scheme of human FGF-1) may be combined with one ormore of the mutation(s) or substitution(s) of “mini-core” residue(s) infurther combination with core packing mutations, such as the following:Phe22→Tyr+Leu44→Phe+Met67→Ile+Leu73→Val+Ala103→Gly+104-106deletion+Phe108→Tyr+Val109→Leu+Leu111→Ile+Cys117→Val+Arg119→Gly+120-122deletion+Phe132→Trp;Ile42→Cys+Leu44→Phe+Met67→Ile+Leu73→Val+Ala103→Gly+104-106deletion+Val109→Leu+Leu111→Ile+Cys117→Val+Arg119→Gly+120-122deletion+Ile130→Cys+Phe132→Trp; orPhe22→Tyr+Ile42→Cys+Leu44→Phe+Met67→Ile+Leu73→Val+Ala103→Gly+104-106deletion+Phe108→Tyr+Val109→Leu+Leu111→Ile+Cys117→Val+Arg119→Gly+120-122deletion+Ile130→Cys+Phe132→Trp. The same mutant combinations may be madeat corresponding positions of mutant FGF proteins having high percentidentity with the 154 and 155 amino acid forms of human FGF-1. Oneskilled in the art would be able to determine the polypeptide sequencesof these mutant FGF proteins based on the disclosure provided herein.

According to some embodiments, the Phe to Trp mutation or substitutionat a position corresponding to position 132 of human FGF-1 (based on the140 amino acid numbering scheme of human FGF-1) may be combined with oneor more mutation(s) or substitution(s) to replace one or moreinteracting N-terminal residues 13 through 17 and/or C-terminal residues131 through 135 (using the 140 amino acid numbering scheme for humanFGF-1) to increase the thermostability of the mutant FGF protein. Forexample, the Phe to Trp mutation or substitution at position 132 (basedon the 140 amino acid numbering scheme of human FGF-1) may be combinedwith one or more of the following mutation(s) or substitution(s) ofthese N-terminal and/or C-terminal residues: Lys12→Cys; Lys12→Thr;Lys12→Val; Leu46→Val; Glu87→Val; Asn95→Val; Pro134→Cys; Pro134→Thr;and/or Pro134→Val. For example, the Phe to Trp mutation or substitutionat position 132 (based on the 140 amino acid numbering scheme of humanFGF-1) may be combined with one or more of the mutation(s) orsubstitution(s) of N-terminal and/or C-terminal residue(s), which may becombined with mutations at symmetry related positions, such as thefollowing: Lys12→Val+Phe132→Trp+Pro134→Val;Lys12→Val+Asn95→Val+Phe132→Trp; Leu46→Val+Phe132→Trp+Pro134→Val;Glu87→Val+Phe132→Trp+Pro134→Val;Leu46→Val+Glu87→Val+Phe132→Trp+Pro134→Val; orLys12→Val+Leu46→Val+Glu87→Val+Asn95→Val+Phe132→Trp+Pro134→Val. The samemutant combinations may be made at corresponding positions of mutant FGFproteins having high percent identity with the 154 and 155 amino acidforms of human FGF-1. One skilled in the art would be able to determinethe polypeptide sequences of these mutant FGF proteins based on thedisclosure provided herein.

According to some embodiments of the present invention, the Phe to Trpmutation or substitution at a position of the mutant FGF proteincorresponding position 132 of human FGF-1 (based on the 140 amino acidnumbering scheme of human FGF-1) may be combined with two or moremutations or substitutions of different types. For example, the Phe toTrp mutation or substitution at a position of the mutant FGF proteincorresponding to position 132 of human FGF-1 (based on the 140 aminoacid numbering scheme of human FGF-1) may be combined with anycombination of mutation or substitution of the following residue types:free cysteine residues, core packing residues, “mini-core” residues,and/or interacting N-terminal or C-terminal residues. Indeed, the Phe toTrp mutation or substitution at a position of the mutant FGF proteincorresponding to position 132 of human FGF-1 (based on the 140 aminoacid numbering scheme of human FGF-1) may be combined with anycombination of mutation(s), modification(s), substitution(s),deletion(s), etc., known in the art or described herein. For example,the following mutant combinations are provided according to someembodiments (based on the 140 amino acid numbering scheme of humanFGF-1): Cys83→Thr+Cys117→Val+Phe132→Trp;Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp;Lys12→Val+Cys117→Val+Phe132→Trp; orLys12→Val+Cys83→Thr+Cys117→Val+Phe132→Trp.

According to some embodiments of the present invention, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 90% identical to the 140 amino acidpolypeptide sequence of wild-type human FGF-1 protein (SEQ ID NO: 1) ora functional fragment thereof, wherein the phenylalanine (Phe) at anamino acid position of the mutant FGF protein corresponding to aminoacid position 132 of wild-type human FGF-1 is replaced with tryptophan(Trp), and wherein the alanine (Ala) at an amino acid position of themutant FGF protein corresponding to amino acid position 66 of wild-typehuman FGF-1 is replaced with cysteine (Cys) with amino acid positionsbased on the 140 amino acid numbering scheme of human FGF-1. Forexample, a mutant FGF protein may have the polypeptide sequence of SEQID NO: 9, or a functional fragment thereof. According to some of theseembodiments, a mutant FGF protein having mutations or substitutionscorresponding to both Ala66→Cys and Phe132→Trp may also have anycombination of one or more other mutation(s), modification(s),substitution(s), deletion(s), etc. known in the art or described herein,such as mutation(s) or substitution(s) of one or more free cysteineresidues, core packing residues, “mini-core” residues, and/orinteracting N-terminal or C-terminal residues.

Most FGFs mediate their biological responses as extracellular proteinsby binding to and activating cell surface tyrosine kinase FGF receptors(FGFRs). Four Fgfr genes, Fgfr1 through Fgfr4, have been identified inhumans and mice, and these may be alternatively spliced to produce agreater number of FGFR isoforms. Except for FGF-11 through FGF-14,FGF-15/19, FGF-21, and FGF-23, other FGFs activate FGFRs with highaffinity and with different degrees of specificity. See, e.g., Itoh, N.et al. (2008), supra. FGF-1 is the only known wild-type mouse/human FGFprotein that is believed to bind to all FGFR types, but other FGFproteins have been shown to bind to multiple FGFRs. See, e.g., Zhang, X.et al., “Receptor Specificity of the Fibroblast Growth Factor Family,” JBiol Chem 281(23):15694-15700 (2006); and Szlachcic et al. (2009),supra, the entire contents and disclosure of which are herebyincorporated by reference. Therefore, a mutant FGF protein according toembodiments of the present invention may bind with specificity andaffinity to at least one FGFR present on the surface of a cell, such asa fibroblast cell, neuronal or neuroblast cell, endothelial cell,chondrocyte, osteoblast, myoblast, smooth muscle cell, or glial cell. Amutant FGF protein according to embodiments of the present invention mayalso trigger growth, proliferation, and/or survival of cells, such asfibroblast cells, neuronal or neuroblast cells, endothelial cells,chondrocytes, osteoblasts, myoblasts, smooth muscle cells, glial cells,etc., known to express one or more FGFRs. This may occur through bindingand activation of a FGF receptor and downstream signaling within thecell.

Both FGF-1 and FGF-2 are members of the FGF-1 or FGF A subfamily andtherefore are more related to each other than any other FGF protein.Human Fgf-2 mRNA encodes a 155 amino acid protein (SEQ ID NO: 4). (SeeFIG. 4) Although FGF-1 is the only FGF protein that has been shown tobind all FGF receptor types, FGF-2 also binds multiple FGF receptortypes and may have therapeutic benefits. Therefore, according to anotherbroad aspect of the present invention, a mutant FGF protein is providedhaving a polypeptide sequence that is at least 90% identical to the 155amino acid polypeptide sequence of wild-type human FGF-2 protein (SEQ IDNO: 4), or a functional fragment thereof, and having one or morecorresponding mutations described above for FGF-1.

According to some embodiments, a mutant fibroblast growth factor (FGF)protein is provided having a polypeptide sequence that is at least 90%identical to the 155 amino acid polypeptide sequence of wild-type humanFGF-2 protein (SEQ ID NO: 4), or a functional fragment thereof, whereinthe alanine (Ala) at an amino acid position of the mutant FGF proteincorresponding to amino acid position 84 of wild-type human FGF-2 isreplaced with cysteine (Cys). Alternatively, a mutant fibroblast growthfactor (FGF) protein is provided having a polypeptide sequence that isat least 95% identical to the 155 amino acid polypeptide sequence ofwild-type human FGF-2 protein (SEQ ID NO: 4), or a functional fragmentthereof, wherein the alanine (Ala) at an amino acid position of themutant FGF protein corresponding to amino acid position 84 of wild-typehuman FGF-2 is replaced with cysteine (Cys). For example, according tosome embodiments, a mutant FGF protein may be SEQ ID NO: 6 or afunctional fragment thereof.

According to some embodiments, a mutant fibroblast growth factor (FGF)protein is provided having a polypeptide sequence that is at least 90%identical to the 155 amino acid polypeptide sequence of wild-type humanFGF-2 protein (SEQ ID NO: 4), or a functional fragment thereof, whereinthe phenylalanine (Phe) at an amino acid position of the mutant FGFprotein corresponding to amino acid position 148 of wild-type humanFGF-2 is replaced with tryptophan (Trp). Alternatively, a mutantfibroblast growth factor (FGF) protein is provided having a polypeptidesequence that is at least 95% identical to the 155 amino acidpolypeptide sequence of wild-type human FGF-2 protein (SEQ ID NO: 4), ora functional fragment thereof, wherein the phenylalanine (Phe) at anamino acid position of the mutant FGF protein corresponding to aminoacid position 148 of wild-type human FGF-2 is replaced with tryptophan(Trp). For example, according to some embodiments, a mutant FGF proteinmay be SEQ ID NO: 8 or a functional fragment thereof.

According to some embodiments of the present invention, a mutant FGFprotein having either the alanine (Ala) to cysteine (Cys) mutation orsubstitution at an amino acid position of the mutant FGF proteincorresponding to amino acid position 84 of the 155 amino acid wild-typehuman FGF-2 (SEQ ID NO: 4), or the phenylalanine (Phe) to tryptophan(Trp) mutation or substitution corresponding to amino acid position 148of the 155 amino acid wild-type human FGF-2 (SEQ ID NO: 4) may becombined with one or more other known mutation(s), substitution(s),modification(s), deletion(s), etc. described herein or known in the art,such as mutation(s) or substitution(s) of one or more free cysteineresidues, core packing residues, “mini-core” residues, and/orinteracting N-terminal or C-terminal residues.

According to some embodiments, a mutant fibroblast growth factor (FGF)protein is provided having a polypeptide sequence that is at least 90%identical to the 155 amino acid polypeptide sequence of wild-type humanFGF-2 protein (SEQ ID NO: 4), or a functional fragment thereof, whereinthe alanine (Ala) at an amino acid position of the mutant FGF proteincorresponding to amino acid position 84 of wild-type human FGF-2 isreplaced with cysteine (Cys), and wherein the phenylalanine (Phe) at anamino acid position of the mutant FGF protein corresponding to aminoacid position 148 of wild-type human FGF-2 is replaced with tryptophan(Trp). For example, a mutant FGF protein may have the polypeptidesequence of SEQ ID NO: 10, or a functional fragment thereof.

As stated above, both FGF-1 and FGF-2 are members of the Fgf-1 or FGF Asubfamily and are therefore more related to each other than any otherFGF protein. Sequence analysis shows that members of different FGFsubfamilies share roughly 15% to 71% amino acid identity, whileorthologous FGF proteins (i.e., members of the same subfamily fromdifferent species) have higher sequence identity. See, e.g., Ornitz etal. (2001), supra; and Itoh et al. (2008), supra. In addition to thewild-type sequences for human FGF-1 and FGF-2 proteins, the wild-typepolypeptide sequences of mouse FGF-1 protein (see FIG. 3; SEQ ID NO: 11)and mouse FGF-2 protein (see FIG. 5; SEQ ID NO: 12) are also provided.Therefore, a mutant FGF protein according to embodiments of the presentinvention having at least 90% or 95% identity to the wild-typepolypeptide sequence of one of the human FGF-1 protein forms is unlikelyto include proteins other than those resembling FGF-1 since members ofother FGF subfamilies generally have much lower sequence identity.Similarly, a mutant FGF protein according to embodiments of the presentinvention having at least 90% or 95% identity to the wild-typepolypeptide sequence human FGF-2 is unlikely to include proteins otherthan those resembling FGF-2 since members of other FGF subfamiliesgenerally have much lower sequence identity.

According to some embodiments of the present invention, the mutant FGFprotein retains the ability to bind with specificity and affinity to aFGF receptor (FGFR) and trigger growth, proliferation, and/or survivalof cultured and/or in vivo cells relative to untreated control cells(i.e., cells that are not exposed to a mutant FGF protein). Such cellsmay include, for example, fibroblast cells, neuronal or neuroblastcells, endothelial cells, chondrocytes, osteoblasts, myoblasts, smoothmuscle cells, glial cells, etc., of human or animal origin known in theart to express one or more FGFRs or to respond to FGF proteins. See,e.g., Esch, F. et al., “Primary structure of bovine pituitary basicfibroblast growth factor (FGF) and comparison with the amino-terminalsequence of bovine brain acidic FGF,” PNAS USA 82(19):6507-11 (1985);and Gensburger, C. et al., “Effect of basic FGF on the proliferation ofrat neuroblasts in culture,” C R Acad Sci III 303(11):465-468 (1986).Such a trigger of growth, proliferation, and/or survival of thesecultured or in vivo cells may occur through binding and activation of aFGF receptor and downstream signaling within the cell(s). According tosome embodiments, the mutant FGF protein of the present invention has agreater thermodynamic stability than wild-type human FGF-1 or FGF-2. Themutant FGF protein of the present invention may have a greaterthermodynamic stability than wild-type human FGF-1 or FGF-2 if themutant FGF protein has a ΔG_(unfolding) value greater than that ofwild-type human FGF-1 or FGF-2, such that ΔΔG=ΔG_(unfolding)(wildtype)−ΔG_(unfolding)(mutant)<0, according to an isothermal equilibriumdenaturation assay, differential scanning calorimetry assay,thermally-monitored spectroscopic assay, or other method of quantitationof ΔG_(unfolding) known in the art. According to some embodiments, themutant FGF protein of the present invention has a greater functionalhalf-life than wild-type human FGF-1 or FGF-2 according to a culturedfibroblast proliferation assay. According to some embodiments, themutant FGF protein of the present invention has essentially unalteredsurface features relative to the wild type FGF protein and therefore haslittle or no immunogenic potential (i.e., the mutant FGF protein doesnot cause a significant immune reaction when introduced or administeredto the body of an individual, subject, or patient).

According to some embodiments of the present invention, a functionalfragment of a mutant FGF protein is also provided, such as a functionalfragment of a polypeptide sequence having at least 90% or 95% identityto at least a portion of the wild-type polypeptide sequence of one ofthe human FGF-1 protein forms or to at least a portion of the wild-typepolypeptide sequence of human FGF-2 protein. A functional fragment maybe defined as a portion or fragment of a mutant FGF protein that retainsFGF-like function. For example, a functional fragment may be a portionor fragment of a mutant FGF protein that is able to bind withspecificity and affinity to at least one FGF receptor (FGFR) present onthe surface of a cell. A functional fragment may also be a portion orfragment of a mutant FGF protein that is able to trigger growth,proliferation, and/or survival of cultured and/or in vivo cells relativeto untreated control cells (i.e., cells that are not exposed to a mutantFGF protein). Such cells may include, for example, fibroblast cells,neuronal or neuroblast cells, endothelial cells, chondrocytes,osteoblasts, myoblasts, smooth muscle cells, glial cells, etc., of humanor animal origin known in the art to express one or more FGFRs orrespond to FGF proteins, which may occur through binding and activationof a FGF receptor and downstream signaling within the cell. The bindingaffinity and/or mitogenicity of a mutant FGF protein, or a functionalfragment thereof, may be determined according to assays described hereinor known in the art, such as a cultured fibroblast proliferation assay,etc.

According to some embodiments, a mutant FGF protein or a functionalfragment thereof that is described herein may further include anyadditional non-FGF peptide sequence or tag known in the art, which maybe used to facilitate its detection or purification. For example, amutant FGF protein may contain any additional non-FGF peptide sequenceknown in the art that provides an epitope or fluorescence for detection,such as Myc, HA, His, FLAG, GST, GFP, etc., or provides a basis forpurification by chromatography. According to some embodiments, a mutantFGF protein or a functional fragment thereof that is described hereinmay further include an additional non-FGF peptide sequence or tag knownin the art for targeting the mutant FGF protein to a particular tissueor cell, improved solubility, sustained activity or stability, improvedexpression, etc.

According to another broad aspect of the present invention, apolynucleotide sequence encoding a mutant FGF protein, or a functionalfragment thereof, of the present invention as described herein is alsoprovided. Wild-type genomic and cDNA polynucleotide sequences for Fgffamily members are known in the art. One skilled in the art would beable to determine the polynucleotide sequences encoding the mutant FGFproteins of the present invention by mutating specific base pairs ofcodon(s) of the wild-type polynucleotide sequence encoding the mutatedor substituted amino acids of the mutant FGF proteins. Such apolynucleotide sequence may be present in (i.e., inserted, introduced,or subcloned into) any vector or expression system known in the art,such as a polynucleotide sequence of a plasmid, artificial chromosome,virus, retrovirus, transposable element, etc. In addition, such a vectoror expression system may be introduced into a host cell, such as abacterial cell, yeast cell, insect cell, mammalian cell, etc., bytransformation, transfection, infection, transduction, direct injection,etc., to allow for expression of the mutant FGF protein, or a functionalfragment thereof, by the host cell. Alternatively, a mutant FGF protein,or a functional fragment thereof, may be synthesized from such apolynucleotide sequence in vitro. Examples of vectors and expressionsystems as well as host cells that may be used for protein expressionare known in the art. See, e.g., U.S. Utility application Ser. No.12/163,755, filed Jun. 27, 2008, the entire contents and disclosure ofwhich are hereby incorporated by reference.

Since a mutant FGF protein, or a functional fragment thereof, accordingto some embodiments of the present invention, includes only polypeptidesequences containing one or more mutations relative to a wild-type FGFsequence, such a mutant FGF protein is not the naturally occurringwild-type polypeptide sequence for the FGF protein. Similarly, apolynucleotide sequence encoding a mutant FGF protein, or a functionalfragment thereof, is not the naturally occurring polynucleotide sequenceencoding the wild-type FGF protein. However, according to someembodiments, a mutant FGF protein, or a functional fragment thereof, ora polynucleotide encoding a mutant FGF protein, or a functional fragmentthereof, may be isolated and/or purified from a cellular or tissueenvironment. For example, a mutant FGF protein, or a functional fragmentthereof, may be isolated and/or purified from a cellular or tissueenvironment from which the mutant FGF protein, or a functional fragmentthereof, is expressed.

According to embodiments of the present invention, novel mutant forms ofFGF family proteins are provided having an increased thermodynamicstability and/or functional half-life. However, it is envisioned thatother mutant forms of FGF family proteins, or a functional fragmentthereof, having instead a decreased thermodynamic stability and/orfunctional half-life may be used, especially when a short half-life ofactivity arid/or rapid clearance is preferred.

According to another broad aspect of the present invention, a mutant FGFprotein, or a functional fragment thereof, may be formulated as part ofa composition containing other components. According to some embodimentsof the present invention, a pharmaceutical composition is providedcomprising a mutant FGF protein, or a functional fragment thereof, incombination with a pharmaceutically acceptable carrier. Such apharmaceutical composition may be administered to an individual,subject, or patient to promote vascularization, healing, proliferation,growth, or protection of cells, etc. According to some embodiments, apharmaceutical composition is provided comprising a therapeuticallyeffective amount of a mutant FGF protein, or a functional fragmentthereof, in combination with a pharmaceutically acceptable carrier.According to some embodiments, for example, a pharmaceutical compositionis provided comprising a mutant FGF protein, or a functional fragmentthereof, together with other angiogenic factors known in the art orprovided herein, such as vascular endothelial growth factor (VEGF),other wild-type and/or mutant FGF proteins, vasodilating molecules,etc., and a pharmaceutically acceptable carrier.

Examples of pharmaceutically acceptable carriers and other suitableadditives and adjuvants for pharmaceutical compositions that may be usedin combination with embodiments of the compounds or compositions of thepresent invention for administration to an individual, subject, orpatient include those known to those skilled in the pharmacological orpharmaceutical arts for use with protein-based biopharmaceuticals. Asused herein, the pharmaceutically acceptable carriers may be eitherliquid or solid and may include solvents, buffers, dispersion media,oils, coatings, surfactants, antioxidants, preservatives (e.g.,antibacterial agents, antifungal agents, etc.), isotonic agents,absorption delaying agents, proteins and low molecular weightpolypeptides, hydrophilic polymers, amino acids, carbohydrates, sugaralcohols, metal ions, salts, preservatives, stabilizers, gels, binders,excipients, fillers, diluents, solubilizers, disintegration agents,lubricants, surfactants, penetrants, chelating agents, sweeteningagents, flavoring agents, dyes, glidants, wetting agents, bulkingagents, thickening agents, etc., and combinations thereof. Examples ofpharmaceutically acceptable carriers may include, for example,substances for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, rate ofdiffusion, odor of the formulation, etc. Since compositions of thepresent invention comprise a mutant FGF protein, pharmaceuticalcompositions may be formulated to include substances that may inhibit oravoid proteolytic degradation.

According to some embodiments of present compositions, the exactformulation, route of administration, and dosage of the presentcompositions comprising a mutant FGF protein may be chosen according tothe judgment of a skilled scientist, veterinarian, or physician in viewof the characteristics and conditions of an individual, subject orpatient to be treated. Proper formulation and choice of pharmaceuticallyacceptable carriers for a pharmaceutical composition may also bedependent upon the route and method of administration. Accordingly,there is a wide variety of suitable formulations for pharmaceuticalcompositions of the present invention. For a description ofpharmaceutical compositions, carriers, formulations, methods and routesof administration, etc., that may be used for embodiments ofcompositions of the present invention, see, for example, Remington, TheScience and Practice of Pharmacy, (University of the Sciences inPhiladelphia, 21st ed., Lippincott Williams & Wilkins, 2005), thecontents and disclosure of which are hereby incorporated by reference.

Except insofar as any conventional pharmaceutical carrier isincompatible with embodiments of the compositions of the presentinvention comprising a mutant FGF protein, their potential use inpharmaceutical compositions of the present invention is contemplated.Embodiments of the pharmaceutical compositions and formulations of thepresent invention may utilize different types of carriers depending onwhether they are to be administered in solid, semi-solid, or liquid formand whether they need to be sterile for certain routes ofadministration, such as local or systemic injection or infusion.

Various delivery systems and reagents known in the art are alsocontemplated for use as carriers for embodiments of pharmaceuticalcompositions of the present invention comprising a mutant FGF protein.Where appropriate, such delivery systems or reagents may include, forexample, liposomes, microparticles or nanoparticles, microcapsules,emulsions, polymers, etc., or any combination thereof. Liposomes may becoated, for example, with opsonization-inhibiting moieties or molecules(e.g., PEG) to avoid detection by the immune system and may bespecifically formulated and/or associated with other molecules,antibodies, or conjugates to improve delivery, intake, and/orspecificity into specific tissues or cells. See, e.g., Szoka et al.,“Comparative properties and methods of preparation of lipid vesicles(liposomes),” Ann. Rev. Biophys. Bioeng., 9:467 (1980); Immordino, M.L., “Stealth liposomes: review of the basic science, rationale, andclinical applications, existing and potential,” Int. J Nanomedicine1(3):297-315 (2006); Samad, A., “Liposomal Drug Delivery Systems: AnUpdate Review,” Current Drug Delivery 4(4): 297-305 (2007), the contentsand disclosures of which are hereby incorporated by reference in theirentirety.

Embodiments of compositions of the present invention may be formulatedso as to provide rapid, sustained, or delayed release of a mutant FGFprotein by embedding or soaking the mutant FGF protein in a matrix ornetwork of polymeric material according to methods known in the art.Embodiments of these compositions may be formulated to restrictdiffusion of a mutant FGF protein away from a location where thecomposition is intentionally administered or applied, such as bydiffusion or by erosion or degradation of the network or matrix. Forlocal administration of embodiments of compositions of the presentinvention, an advantage of providing sustained or restricted release isthat a localized efficacious concentration of a mutant FGF protein maybe achieved at a site of administration with relatively less mutant FGFprotein and fewer applications or injections required. Such a restrictedor sustained release composition may provide targeted delivery of amutant FGF protein while minimizing undesired side effects that mayresult if the mutant FGF protein diffused away from the site ofadministration.

Embodiments of compositions of the present invention providing sustainedor restricted release or diffusion of a mutant FGF protein may include avariety of biocompatible materials or polymers, such aspoly(2-hydroxyethyl methacrylate), ethylene vinyl acetate orpoly-D-(−)-3-hydroxybutyric acid, polylactides, polyglycolides,polylactide co-glycolide, polyanhydrides, poly(ortho)esters,polypeptides, hyaluronic acid, hydrogels, collagen, fibrin, alginate,chondroitin sulfate, carboxylic acids, fatty acids, phospholipids,polysaccharides, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone, sulfated proteoglycans, dextrins, poloxamers,silicone, methylcellulose, and the like. Such compositions may comprisea semi-permeable polymer matrix or network, such as a gel, paste, putty,etc. According to some embodiments, compositions may be molded or formedinto a desired shape, such as for placement or to fill a space atdesired site of administration in the body of an individual, subject, orpatient to promote vascularization, cellular proliferation, and/orhealing, or to promote specific routes or tracks of novel vasculature.

According to some embodiments of the present invention, a pharmaceuticalcomposition of the present invention comprising a mutant FGF protein mayinclude a matrix or network containing one or more of the following:collagen, fibrin, fibrinogen, fibronectin, and/or alginate. For example,a pharmaceutical composition of the present invention comprising amutant FGF protein may be formulated as a fibrin plug or fibrin glue.According to other embodiments, compositions of the present inventioncomprising a mutant FGF protein may be embedded or soaked into a medicalor surgical device, such as a fabric, bandage, suture, sponge, etc. orother polymers, which may safely degrade over time.

The mode or route of administration for embodiments of compositions ofthe present invention comprising a mutant FGF protein may be selected tomaximize delivery to a desired target site in the body of an individual,subject, or patient. Pharmaceutical compositions may be administered ina number of ways, including any suitable enteral, parenteral, topical,or local mode or route, depending on whether local or systemic treatmentis preferred and/or the specific area to be treated. Suitable enteralroutes for administration may include oral, rectal, intestinal, andgastric. Suitable parenteral routes may include intravascular routes,such as intravenous (bolus and infusion), intraarterial, andintracardiac; mucosal routes, such as transmucosal (e.g., insufflation),sublingual, buccal, intranasal, pulmonary (e.g., inhalation), andvaginal; intracranial (e.g., intracerebral); intraocular; intrathecal;intraperitoneal; subcutaneous; intramuscular; intradermal; subcutaneous;intramedullary; intraarticular; or intraosseus.

Embodiments of pharmaceutical compositions of the present invention maybe administered in a variety of unit dosage forms depending on themethod of administration. For example, unit dosage forms suitable fororal administration include solid dosage forms, such as powders,granules, tablets, pills, capsules, suppositories, depots, and dragees,and liquid dosage forms, such as elixirs, syrups, suspensions, sprays,gels, lotions, creams, slurries, foams, jellies, ointments, salves,solutions, suspensions, tinctures, and/or emulsions.

Embodiments of pharmaceutical compositions of the present invention maybe administered either locally or systemically. However, polypeptidesare generally less suitable for oral administration due to theirsusceptibility to digestion by gastric acids or intestinal enzymes.Although formulations may be designed to circumvent these problems,bioavailability is impaired due to poor gastrointestinal absorption.Therefore, preferred routes of administration generally includeparenteral administration or local injection or topical application ator near a site of a disorder or disease, such as a site of ischemic orhypoxic stress or a site of an injury or wound.

Suitable routes for parenteral administration are described above.Embodiment of pharmaceutical compositions comprising a mutant FGFprotein for parenteral administration may be formulated as solutions,emulsions, suspensions, or other liquids, such as saline, dextrosesolution, glycerol, and the like, which may be sterile and/or isotonic.However, a suitable carrier for parenteral administration may includeaqueous or non-aqueous (e.g., oily) solvents. Suitable formulations forparenteral administration may be in unit-dose or multi-dose sealedcontainers, such as ampoules, vials, bags, etc. A mutant FGF protein ofthe present invention may be administered by continuous infusion (e.g.,minipumps, osmotic pumps, etc.), single bolus, or slow-release depotformulations, etc. Solutions and suspensions for parenteraladministration may be freshly prepared or resuspended from a drypreparation of a mutant FGF protein, such as a lyophilized or spraydried preparation, prior to its use.

In addition to parenteral modes of administration, embodiments ofpharmaceutical compositions comprising a mutant FGF protein may beadministered by local injection, placement, catheter delivery, orimplantation at a desired site of action in the body of an individual,subject, or patient, such as a tissue or cellular environment at or neara site of a disorder or disease, such as at or near a site of ischemicor hypoxic stress or a site of an injury or wound. Alternatively, apharmaceutical composition comprising a mutant FGF protein may beadministered by local injection, placement, or implantation at or near asite causing an ischemic or hypoxic stress or condition at a differentsite, such as a site of vessel occlusion. Such a local injection,placement, or implantation of pharmaceutical compositions of the presentinvention may include any suitable peri- and intra-tissue injections,such as intradermal, intramuscular, intracardiac, subcutaneous,intrathecal, etc. as the case may be. Pharmaceutical compositions of thepresent invention may also be administered by local injection,placement, or implantation at two or more sites at or near a desiredsite of action.

For local injection, placement, or implantation, embodiments ofpharmaceutical compositions of the present invention may be formulatedwith a variety of aqueous or non-aqueous solutions, suspensions,emulsions, etc. as described above, such as physiologically compatiblebuffers including Hank's solution, Ringer's solution, physiologicalsaline buffer, etc. Embodiments of pharmaceutical compositions for localinjection, placement, or implantation may also comprise biocompatiblematerials or polymers providing sustained release or restricteddiffusion as described above. As with pharmaceutical compositions forparenteral administration, solutions and suspensions for local ortopical administration may be freshly prepared or resuspended from a drypreparation of a mutant FGF protein, such as a lyophilized or spraydried preparation, prior to its use.

Embodiments of pharmaceutical compositions of the present invention mayalso be administered topically, such as at a site of a tissue injury ora wound. Embodiments of pharmaceutical compositions for topicaladministration may be formulated as a liquid or semi-solid material,such as a gel, paste, putty, ointment, cream, emulsion, patch, etc. aswell as other biocompatible materials or polymers. However, embodimentsof pharmaceutical compositions for topical administration may also beformulated as a dry or solid preparation, such as a powders, granules,etc., that may be applied directly to a desired site of action.According to some embodiments, pharmaceutical compositions for topicaladministration may be molded into a desired size and shape, such as forplacement within or to fill a space at a desired site of administrationin the body of an individual, subject, or patient, such as at a site ofa tissue injury or wound to promote healing. Pharmaceutical compositionsof the present invention may also be topically administered at two ormore sites at or near a tissue injury or a wound.

Such embodiments of pharmaceutical compositions of the present inventioncomprising a mutant FGF protein for topical or local administration maycomprise a semi-permeable polymer matrix or network, which may providesustained release or restricted diffusion to localize the mutant FGFprotein to the site of administration. According to some embodiments ofthe present invention, a pharmaceutical composition of the presentinvention comprising a mutant FGF protein for topical or localadministration may include a matrix or network of fibrin and/orfibrinogen, such as a fibrin plug or fibrin glue.

Determination of a therapeutically effective amount of a mutant FGFprotein, or a functional fragment thereof, may be carried out in amanner known to those skilled in the art depending on the conditions orexigencies of a given therapeutic situation. For example, atherapeutically effective amount may be determined by titration tooptimize safety and effectiveness. Lower than expected dosages may beadministered first to an individual, subject, or patient, and dosagesmay then be titrated upward until a therapeutically effective and safeconcentration amount (or a potentially unsafe concentration or amount)is reached.

Appropriate dosage amounts for a mutant FGF protein may be determined orpredicted from empirical evidence. Specific dosages may vary accordingto numerous factors and may be initially determined on the basis of invitro, cell culture, and/or animal in vivo studies. Dosages orconcentrations tested in vitro for mutant FGF proteins according to someembodiments of the present invention may provide useful guidance indetermining therapeutically effective and appropriate amounts for invivo administration. For example, a therapeutically effective dose of amutant FGF protein may be estimated initially from a cell culture assay,for example, by measuring proliferation, growth, and/or survival ofcultured cells or by the formation of vessels in culture in response tothe mutant FGF protein depending on the intended therapeuticapplication. Such values may be used, for example, to translate intoappropriate amounts for use in animal testing or for clinical trials inhumans. Determining an appropriate dosage for composition according toembodiments of the present invention may be discerned from any or allinformation or data available from any assay or experiment performed.

Animal testing of predicted dosages may provide additional indication ofproper dosages for other types of animals, including humans. Forexample, a dosage for a mutant FGF protein may be an amount thatproduces a localized or circulating concentration which roughlyapproximates concentrations shown to be effective according to cellculture and/or in vitro assays. Such an amount may be used initially todetermine effectiveness and/or safety at such concentrations or todetermine or extrapolate useful dosages for other animals, such ashumans. Toxicity and therapeutic efficacy of a mutant FGF protein may bedetermined or predicted from any standard pharmaceutical proceduresbased on data from cell cultures or experimental animals. For example,an LD₅₀ value (i.e., dose lethal in 50% of the population) and an ED₅₀value (the dose therapeutically effective in 50% of subjects accordingto clinical or pathological criteria) may be determined for a givenanimal test subject, and the ratio of LD₅₀/ED₅₀ may be expressed as atherapeutic index for a parenterally administered mutant FGF protein.Compounds that exhibit a high therapeutic index may indicate that higherconcentrations of the mutant FGF protein are safe and non-toxic and/orthat lower doses may be efficacious in an individual, subject, orpatient. However, a lower therapeutic index might indicate that onlylower (and perhaps ineffective) concentrations of the mutant FGF proteinmay be acceptable in terms of safety. Levels of mutant FGF protein in atissue or plasma sample from an individual, subject, or patient may bemeasured or monitored by any known technique.

In most cases, an appropriate dosage amount may be a balance of factorsincluding efficacy and safety. Factors considered in determining adosage that is therapeutically effective and safe for an individual,subject, or patient in clinical settings will depend on many factorsincluding the mode/route of administration, timing of administration,rate of excretion, target site, disease or physiological state, medicalhistory, age, sex, physical characteristics, other medications, etc.This list of factors is illustrative and not exhaustive, and may includeany or all factors which might be considered by a skilled scientist,veterinarian, or physician (as the case may be) in determining anappropriate treatment. A specific dosage amount of a mutant FGF proteinadministered to an individual, subject, or patient may be in a rangeequivalent to dosages used for other currently-used therapeuticproteins, adjusted for the altered activity, thermostability, orfunctional half-life of the particular mutant FGF protein.

For the purposes of the present invention, a therapeutically effectiveamount of a mutant FGF protein may refer to an amount effective toachieve a desired result, purpose, or therapeutic benefit, such as anamount effective to prevent, alleviate, ameliorate, treat, etc. theunderlying causes and/or symptoms of a condition or disease, such as anischemic or hypoxic condition or disease or a wound or tissue damage.According to some embodiments, a therapeutically effective amount of amutant FGF protein may be an amount effective to increase blood flow,angiogenesis, and/or vascularization within or to a particular tissue orregion of the body of an individual, subject, or patient, such as atissue or region of the body experiencing ischemia and/or hypoxicconditions. Increased blood flow, angiogenesis, and/or vascularizationwithin or to such a tissue or region of the body may be determined by askilled scientist, veterinarian, or physician using any known reagentsand pathological or clinical techniques, such as imaging techniquesusing a contrast dye to detect vasculature, reduction of clinicalsymptoms associated with an underlying ischemic or hypoxic condition ordisease, etc. Where a mutant FGF protein is administered to cardiactissue, a therapeutically effective amount may be an amount effective toreduce clinical symptoms of coronary artery disease, such as reductionin angina, breathlessness, leg swelling, heart or respiratory rates,edema, fatigue, weakness, etc., or to reduce the risk of a myocardialinfarction. According to other embodiments, a therapeutically effectiveamount of a mutant FGF protein may be an amount effective to improve thequality and/or rate of healing or repair of a damaged tissue or woundaccording to known standards and knowledge generally available to askilled scientist, veterinarian, or physician as the case may be.

Therapeutically effective amounts or dosages of a mutant FGF protein mayinclude any dosage amounts, or approximations thereof, of a FGF proteinpreviously used or contemplated for use in treatments or clinicaltrials, or extrapolated from dosage amounts used in experimentalanimals. For example, a therapeutically effective amount may be a dosageof about 0.01 mg per kg body weight. See, e.g., Schumacher, B. et al.,“Induction of neoangiogenesis in ischemic myocardium by human growthfactors: first clinical results of a new treatment of coronary heartdisease,” Circulation 97:645-650 (1998), the entire contents anddisclosure of which is hereby incorporated by reference.

Embodiments of the compounds or compositions of the present inventionmay be administered either as a single dose or as part of a dosageregimen. A dosage regimen may be adjusted to provide an optimumtherapeutic response. For example, several or multiple doses maybe-administered at a predetermined time interval and doses may beproportionally reduced as indicated by the exigencies of a therapeuticsituation. By administering an embodiment of a composition of thepresent invention as part of a dosage regimen, circulating or localconcentrations may be allowed to reach a desired equilibriumconcentration for a compound through a series of doses. Depending on theseverity and responsiveness of the condition to be treated, dosing canalso be a single administration of a slow release composition, withcourse of treatment lasting from several days to several weeks or monthsor until the disease or condition is cured or an effective reduction inthe underlying causes and/or symptoms of the disease state is achieved.

According to another broad aspect of the present invention, methods areprovided for treating, inhibiting, preventing, managing, ameliorating,etc., an ischemic or hypoxic condition or disease (i.e., diseases orconditions caused by insufficient blood flow to one or more tissues),such as coronary artery disease, peripheral vascular occlusion ordisease, peripheral arterial disease (e.g., critical limb ischemia orCLI), avascular necrosis, such as osteonecrosis, aseptic necrosis, orischemic bone necrosis, and the like, by administering a mutant FGFprotein to an individual, subject, or patient. According to someembodiments, a composition comprising a mutant FGF protein may beadministered by local injection, injection or delivery viacatheterization, placement, or implantation at a desired site of actionin the body of an individual, subject, or patient, such as byapplication at, into, onto, or near a site of a disorder or disease(e.g., at, into, onto, or near a tissue or cellular environmentexperiencing ischemic or hypoxic stress).

Alternatively, a pharmaceutical composition comprising a mutant FGFprotein may be administered by local injection, placement, orimplantation at, into, onto, or near a site causing an ischemic orhypoxic stress or condition at a different site, such as at or near asite of vessel occlusion, which may be caused by atherosclerosis orplaque formation. For example, a composition comprising a mutant FGFprotein may be administered at or near one or more sides of a site ofvessel occlusion to encourage new vessels to augment blood flow or tobypass the site of blockage.

Such a local injection, injection or delivery via catheterization,placement, or implantation of pharmaceutical compositions of the presentinvention may include any form of suitable peri- and intra-tissue modesof injection, placement, or implantation, such as intradermal,intramuscular, intracardiac, subcutaneous, intrathecal, etc. as the casemay be. In the case of coronary artery disease, for example, acomposition comprising a mutant FGF protein may be administered by localinjection or placement at, into, onto, or near cardiac tissue or cardiacmuscle.

According to another broad aspect of the present invention, methods areprovided for improving the quality and/or rate of tissue repair and/orwound healing by administering a mutant FGF protein to an individual,subject, or patient. According to some embodiments, a compositioncomprising a mutant FGF protein may be administered by injection,placement, or implantation at, into, onto, or near a site of a wound ortissue damage. According to some embodiments, such a wound or tissuedamage may be caused by traumatic injury. According to otherembodiments, such a wound or tissue damage may be immunologicallymediated. By administering a mutant FGF protein to a site of a wound ortissue damage, wound healing or tissue repair may be promoted orimproved by increased growth, proliferation, and/or survival of cellsand/or angiogenesis to provide blood flow to the repaired or healedtissue. For example, a composition comprising a mutant FGF protein maybe administered according to some embodiments to cardiac or brain tissuefollowing myocardial infarction or stroke, respectively, to promoterepair, neovascularization, and/or healing of the damaged tissues.According to some embodiments, a composition comprising a mutant FGFprotein may be administered by injection, catheterization, placement, orimplantation at, into, onto, or near a site of an incision or tissuedamage or removal resulting, at least in part, from a surgical operationto promote healing and repair of the tissue.

According to some embodiments, a composition comprising a mutant FGFprotein may be administered according to some embodiments to anindividual, subject, or patient having a neural injury due to trauma ordisease. Such a neural injury may be due to an immunologic condition,disorder, or disease, such as transverse myelitis (TM) including acutetransverse myelitis (ATM) or idiopathic transverse myelitis (ITM),brachial plexus injury including obstetric brachial plexus injury(OBPP), or other spinal cord or peripheral nerve injuries or diseases.By administering a composition comprising a mutant FGF protein byinjection, placement, or implantation at, into, onto, or near a site ofneuronal tissue damage in the body of an individual, subject, orpatient, the proliferation, growth, regeneration, and/or survival ofneuronal cells may be achieved to promote repair or healing of neuronaltissue, which may lead to a reduction or alleviation of the causes orsymptoms of neuronal disease or tissue damage.

Following administration of an embodiment of a composition comprising amutant FGF protein, progress against a condition or disease may bemonitored by a skilled scientist, veterinarian, or physician using anyknown or available reagent, assay, pathological or medical technique,equipment, etc. For example, progress against an ischemic or hypoxiccondition or disease may be monitored by determining the amount of bloodflow, vascularization, and tissue oxygenation following treatment.Imaging devices and techniques permit the visualization of vessels andblood flow especially with a contrast dye, and other devices andtechniques, such as oximeters, etc., may be used to measure tissueoxygenation.

According to another broad aspect of the present invention, cells may begrown ex vivo (e.g., in culture following removal from a donorindividual and prior to transfer to a recipient) for use intransplantation, tissue engineering, or engraftment into an individual,subject, or individual receiving the cells as treatment. Growing orculturing cells ex vivo may allow for their expansion and/ormanipulation prior to use. According to these embodiments, a mutant FGFprotein may be introduced to these cells grown ex vivo or in culture toimprove the growth, expansion, proliferation, and/or survival. Accordingto some embodiments, the donor may be the same individual, subject, orpatient as the recipient.

Having described the many embodiments of the present invention indetail, it will be apparent that modifications and variations arepossible without departing from the scope of the invention defined inthe appended claims. Furthermore, it should be appreciated that allexamples in the present disclosure, while illustrating many embodimentsof the invention, are provided as non-limiting examples and are,therefore, not to be taken as limiting the various aspects soillustrated.

EXAMPLES Example 1 Structural Basis of Conserved Cysteine in FGF ProteinFamily

The cysteine residue at position 83 of human FGF-1 is conserved amongthe 22 mouse/human FGF proteins. In this example, the structural role ofCys 83 in FGF-1 as well as the effect of mutation of Cys 83 on proteinstability is examined. In addition, the effects of mutating the alanineat position 66 of human FGF-1 to cysteine are determined.

Materials and Methods

Mutagenesis and Expression: Experiments in this example utilize asynthetic gene for the 140 amino acid form of human FGF-1 containing anadditional amino-terminal His tag as previously described. See, e.g.,Brych et al. (2001), supra; Linemeyer, D. L. et al., “Disulfide bondsare neither required, present, nor compatible with full activity ofhuman recombinant acidic fibroblast growth factor,” Growth Factors3:287-298 (1990); Blaber, M. et al., “X-ray crystal structure of humanacidic fibroblast growth factor,” Biochemistry 35:2086-2094 (1996);Gimenez-Gallego, G. et al., “The complete amino acid sequence of humanbrain-derived acidic fibroblast growth factor,” Biochemical andBiophysical Research Communications 128:611-617 (1986); and Cuevas, P.et al. (1991), supra, the entire contents and disclosure of which arehereby incorporated by reference. The QuikChange™ site directedmutagenesis protocol (Stratagene, La Jolla, Calif.) is used to introducepoint mutations, which may be confirmed by nucleic acid sequenceanalysis (Biomolecular Analysis Synthesis and Sequencing Laboratory,Florida State University). Expression and purification protocols mayfollow previously published procedures. Purified protein is exchangedinto 50 mM sodium phosphate, 0.1 M NaCl, 10 mM ammonium sulfate, 2 mMdithiothreitol (DTT), pH 7.5 (“crystallization buffer”). Due to thepotential for disulfide bond formation, the purified Ala66→Cys mutantprotein is exchanged against crystallization buffer both with andwithout the inclusion of DTT. The yield for most of the mutant proteinsis 20-40 mg/L. However, Cys83→Ile could not be isolated due tosignificant precipitation during purification (suggesting substantialdestabilization). Therefore, Cys83→Ile is constructed in aLys12→Val+Cys117→Val stabilizing background. See, e.g., Dubey, V. K. etal. (2007), supra. Lys12→Val+Cys117→Val is chosen for this example sincethese mutation sites are distal to position Cys83 while providing −8.1kJ/mol of additional thermostability.

Isothermal Equilibrium Denaturation: This method makes use of thefluorescence signal of the single endogenous Trp residue at position107. This residue is ˜90% buried in the native structure and istherefore useful as a spectroscopic probe of protein denaturation.Complete details of the instrumentation, data collection and analysisprocedure have been previously reported. See, e.g., Blaber, S. I. etal., “Reversible thermal denaturation of human FGF-1 induced by lowconcentrations of guanidine hydrochloride,” Biophysical Journal77:470-477 (1999), the entire contents and disclosure of which arehereby incorporated by reference. Briefly, the fluorescence signal ofFGF-1 is atypical in that Trp107 exhibits greater quenching in thenative state rather than the denatured state. Excitation at 295 nmprovides selective excitation of Trp107 in comparison with the six Tyrresidues that are present in the structure. About 5 μM of proteinsamples in various concentrations of GuHCl are allowed to equilibrateovernight at room temperature (298 K). Triplicate scans are collectedand averaged and buffer traces are collected and subsequently subtractedfrom protein scans. Scans are integrated to quantify the totalfluorescence as a function of denaturant concentration. In this example,the data is analyzed using a general purpose non-linear least-squaresfitting program (DataFit, Oakdale Engineering, Oakdale Pa.) implementinga six parameter, two-state model as described by the following equation(1):

$\begin{matrix}{F = \frac{F_{0\; N} + {S_{N}\lbrack D\rbrack} + {\left( {F_{0\; D} + \left( {S_{D}\lbrack D\rbrack} \right)} \right)e^{{- {({{\Delta \; G_{0}} + {m{\lbrack D\rbrack}}})}}/{RT}}}}{1 + e^{{- {({{\Delta \; G_{0}} + {m{\lbrack D\rbrack}}})}}/{RT}}}} & (1)\end{matrix}$

where [D] is the denaturant concentration, F_(0N) and F_(0D) are the 0 Mdenaturant intercepts for the native and denatured state baselines,respectively, and S_(N) and S_(D) are the slopes of the native anddenatured state baselines, respectively. See, e.g., Eftink, M. R. “Theuse of fluorescence methods to monitor unfolding transitions inproteins,” Biophysical Journal 66:482-501 (1994), the entire contentsand disclosure of which are hereby incorporated by reference. ΔG₀ and mdescribe the linear function of the unfolding free energy versusdenaturant concentration, where ΔG₀ refers to ΔG_(unfolding) valueextrapolated to 0M denaturant concentration (i.e. the y-intercept of thelinear function) and m is the slope. In this example, the effect of agiven mutation upon the stability of the protein (ΔΔG) is calculated bytaking the difference between the C_(m) values for wild-type and mutantproteins and multiplying by the average of the m values as described bythe following equation (2):

ΔΔG=(C _(m WT) −C _(m mutant))(m _(WT) +m _(mutant))/2   (2)

where a negative value indicates the mutation is stabilizing inrelationship to the wild type protein. See, e.g., Pace, C. N. et al.,“Measuring the conformational stability of a protein,” ProteinStructure: a Practical Approach, Creighton, T. E., ed., p. 299-321,(Oxford University Press; Oxford, UK 1997), the relevant contents anddisclosure of which are hereby incorporated by reference.

Crystallization, data collection, molecular replacement, and refinement:Purified mutant protein in crystallization buffer is concentrated to9-13 mg/ml, and crystals are grown using the hanging-drop vapordiffusion method. Crystals suitable for diffraction are grown in oneweek at room temperature with 1 ml of reservoir solution containing2.0-3.5 M sodium formate and 0.1-1.0 M ammonium sulfate in thecrystallization buffer. The Ala66→Cys mutant crystal setups areperformed in crystallization buffer in the presence and absence of DTT.Diffraction data for each of the mutant proteins, exceptCys83→Ile+Lys12→Val+Cys117→Val, is collected using a Rigaku RU-H2Rrotating anode X-ray source (Rigaku MSC, The Woodlands, Tex.) equippedwith Osmic Blue confocal mirrors (MarUSA, Evanston, Ill.) and a RigakuR-axis Ile image plate detector. In this example, diffraction ofCys83→Ile+Lys12→Val+Cys117→Val mutation is performed at the SoutheastRegional Collaborative Access Team (SER-CAT) 22-BM beam line (λ=1.00 Å)at the Advanced Photon Source at Argonne National Laboratory using aMarCCD225 detector (MarUSA, Evanston, Ill.). Crystals are mounted usingHampton Research (Aliso Viejo, Calif.) nylon mounted cryo-turns andfrozen in a stream of nitrogen gas at 100 K. Diffraction data isindexed, integrated and scaled using the DENZO software package. See,e.g., Otwinowski, Z., Proceedings of the CCP4 Study Weekend: “DataCollection and Processing,” (1993); and Otwinowski, Z. et al.,“Processing of x-ray diffraction data collected in oscillation mode,”Methods in Enzymology 276:307-326 (1997), the entire contents anddisclosure of which are hereby incorporated by reference. His-taggedwild-type FGF-1 (PDB code: 1JQZ) is used as the search model inmolecular replacement for structures using the Crystallography and NMRSystem (CNS) software. See, e.g., Brunger, A. T. et al.,“Crystallography and NMR system (CNS): A new software system formacromolecular structure determination,” Acta CrystallographicaD54:905-921 (1998), the entire contents and disclosure of which arehereby incorporated by reference. Model building and visualizationutilizes the O molecular graphics program. See, e.g., Johnson, D. E. etal., “The human fibroblast growth factor receptor genes: a commonstructural arrangement underlies the mechanisms for generating receptorforms that differ in their third immunoglobulin domain,” Molecular andCellular Biology 11:4627-4634 (1991), the entire contents and disclosureof which are hereby incorporated by reference. Structure refinementutilizes CNS software, with 5% of the data in the reflection files setaside for R_(free) calculations. Quantification of solvent-excludedcavities with the refined mutant structures is performed using the MSPsoftware package. See, e.g., Connolly, M. L., “The molecular surfacepackage,” Journal of Molecular Graphics 11:139-141 (1993), the entirecontents and disclosure of which are hereby incorporated by reference.

Results

Mutant protein purification: The mutant proteins in this example exceptthe Cys83→Ile and Ala66→Cys mutations are purified with equivalent yield(˜20 mg/L) and purity (i.e., homogeneity by Coomassie Blue stained SDSPAGE) as the wild-type recombinant protein. The Cys83→Ile mutationexhibits very low solubility resulting in substantial precipitationduring purification. However, when constructed in theLys12→Val+Cys117→Val mutant background, theCys83→Ile+Lys12→Val+Cys117→Val mutant is purified with a yield of ˜15mg/L of soluble protein. The Ala66→Cys mutation, exchanged into a finalbuffer with no DTT, yields a doublet band (of varying intensitydepending on preparation) using SDS PAGE under non-reducing conditions.This doublet is resolved as a single band (with migration rateequivalent to the slower-migrating band of the non-reduced doublet) uponreduction. Thus, as initially purified under non-reducing bufferconditions, the Ala66→Cys mutant contains a partially-formedintra-molecular disulfide bond. Homogenous preparations of a reducedform of the Ala66→Cys mutation could readily be achieved by theinclusion of 10 mM DTT in buffer components utilized throughout thepurification.

Isothermal equilibrium denaturation: For this example, the derivedthermodynamic parameters for the mutant proteins in comparison towild-type FGF-1 (or Lys12→Val/Cys117→Val reference mutant) aresummarized in the table in FIG. 6. Point mutations at position 83destabilize the protein and vary from about 5.2 to about 12.6 kJ/mol.ΔΔG for the Cys83→Ser mutation is about 6.8 kJ/mol in good agreementwith our previous report. See Culajay (2000), supra. The proteinsexhibit excellent agreement with a two-state denaturation model, withthe exception of the Ala66→Cys mutation. Under reducing conditions, theisothermal equilibrium denaturation of Ala66→Cys is described by atwo-state process having a denaturation midpoint of about 1.01 M GuHCl.(See table in FIG. 6). However, under conditions where DTT is omittedfrom the purification buffer, the unfolding transition has twodiscernable components with substantially different midpoints ofdenaturation. The relative fraction of each component is variable, anddepends upon the particular preparation. Fully reduced Ala66→Cys mutantprotein is therefore prepared and exchanged into non-reducing buffer(i.e., crystallization buffer with no DTT), and the isothermalequilibrium unfolding behavior is characterized as a function ofincubation time (see FIG. 8). These results show that under airoxidation the initially fully-reduced Ala66→Cys mutant protein slowlyconverts to a more stable form having a denaturation midpoint of about1.87 M GuHCl (see table in FIG. 6). The rate of loss of the reduced formproceeds with apparent first-order exponential kinetics with a half-lifeof about 60 hours (see FIG. 9) and is inversely proportional to theformation of the oxidized form. The emergence of the higher-stabilityoxidized form also corresponds to a faster-migrating band undernon-reducing SDS PAGE, consistent with formation of a specificintra-molecular disulfide bond (see FIG. 9). Analysis of the isothermalequilibrium data associated with the more stable oxidized form showsthat it is 10.4 kJ/mol more stable than the wild-type protein and 13.6kJ/mol more stable than the reduced form (see FIGS. 6 and 10).

X-ray structure determination: Diffraction-quality crystals wereobtained for the Cys83→Ser, Cys83→Thr, Cys83→Ala, Cys83→Val,Cys83→Ile+Lys12→Val+Cys117→Val, reduced and oxidized forms of Ala66→Cysmutant proteins. X-ray diffraction data sets with resolution rangesbetween 1.90 and 2.55 Å are collected with excellent completion in eachcase. Structures are refined to acceptable crystallographic residual andstereochemistry. (See table in FIG. 7) The mutants, exceptCys83→Ile+Lys12→Val+Cys117→Val and reduced Ala66→Cys, are crystallizedin orthorhombic space group (C222₁) with two molecules in the asymmetricunit (isomorphous with the wild-type FGF-1 crystal form). TheCys83→Ile+Lys12→Val+Cys117→Val and reduced Ala66→Cys mutant proteinsboth crystallized in a related P2₁ monoclinic space group. Analysis ofthis P2₁ cell indicated a Matthews' coefficient of 2.8 Å³/Da with fourmolecules in the asymmetric unit. These four molecules are successfullypositioned using molecular replacement with wild-type FGF-1 (PDB ID:1JQZ) as the search model, in both cases. The 2F_(o)−F_(e) differenceelectron density is unambiguous at the mutation site(s), and the mutantstructures could be accurately modeled in each case.

Summaries of individual X-ray structures is provided in the followingparagraphs:

Cys83→Ala: The wild-type Cys83 Sγ participates as an H-bond acceptorwith the main chain amide of residue Asn80 and with an H-bond distanceof 3.5 Å. The Ala mutation deletes the side chain Sγ group and theassociated H-bond with the Asn80 main chain amide (see FIG. 11A). Thestructure of the Ala mutation shows no obvious H-bond partner for theAsn80 main chain amide, and it therefore appears as an unsatisfiedH-bond group. There is minimal structural collapse in response to thedeletion of the side chain Sγ group, and a cavity calculation indicatesthe presence of a novel 7 Å³ cavity (detected using 1.0 Å radius probe)adjacent to the C^(β) at the former location of the Sγ.

Cys83→Ser: The Ser mutation substitutes an Oγ hydroxyl for the wild-typeCys Sγ. The mutant Ser side chain adopts the same rotamer (gauche+) asthe wild-type Cys, and is thus oriented appropriately to participate asan H-bond acceptor to the Asn80 main chain amide. Due to the smallerdiameter of the oxygen atom, the structure must collapse somewhat tobring the Asn80 main chain amide within effective H-bond distance of themutant Ser83 Oγ. However, the mutant structure shows minimal evidence ofany such structural adjustment, and the resulting Ser83 Oγ and Asn80 Natomic distance is 3.7±0.4 Å (average of two independent molecules inthe asymmetric unit) indicating that no significant H-bond interactionoccurs between these groups. The most pronounced structural change inthe Ser mutant is a 20° rotation of the Asn80 γ₂ angle, resulting in anovel H-bond interaction (2.7 Å) between the introduced Ser83 Oγ andAsn80 Oδ1 (see FIG. 11B). In this H-bond interaction, the Ser83 Oγ isthe donor to the Asn80 Oδ1 acceptor. Thus, the mutant Ser side chain isparticipating in a different type of H-bond interaction in comparison tothe wild-type Cys. In adjusting to position its acceptor group towardsthe introduced Ser, the Asn80 Oδ1 breaks its H-bond with the main chainamide at position Glu82. Consequently, this main chain amide is anunsatisfied H-bond group.

Cys83→Thr: The Cγ2 atom of the mutant Thr83 side chain juxtaposes thewild-type Cys Sγ atom (FIG. 11C). In this orientation, the H-bondinteraction with the main chain amide of position Asn80 is lost, andthis amide appears as an unsatisfied H-bond donor. The rotamerorientation of the mutant Thr positions the side chain Oγ1 atom adjacentto the aromatic ring of Tyr55, which moves slightly to avoid close vander Waals contact. In this orientation the introduced Thr Oγ1participates as an H-bond donor to the main chain carbonyl group ofAsn80.

Cys83→Val: The mutant Val side chain rotamer is essentially isosteric tothat of the Thr83 mutant (FIG. 11D). As with the Thr83 mutant, the Asn80main chain amide H-bond is lost. Furthermore, the novel H-bond observedwith the mutant Thr83 Oγ1 and Asn80 main chain carbonyl is not present.Additionally, the movement of the aromatic ring of Tyr55 appears morepronounced than with the Thr mutant.

Cys83→Ile: The mutant Ile side chain Cβ methyl groups are notaccommodated isosteric to the related Val83 mutant. Rather, the Ile Cγ2is oriented towards the aromatic ring of Tyr55 which rotates ˜90% toavoid close van der Waals contact (see FIG. 11E). With this rotamer, themutant Ile Cγ1 juxtaposes with the wild-type Cys83 Sγ, and thispositions the Ile Cδ1 atom between the Tyr64 aromatic ring and the Cα ofresidue position Pro79. The Cα of Pro79 shifts ˜1.0 Å to accommodate theintroduced Ile Cδ1.

Ala66→Cys (reduced): The reduced form of the Ala66→Cys mutant yieldeddiffraction quality crystals. The P2₁ crystal form obtained has fourmolecules in the asymmetric unit. In two of these, Cys83 adopts thewild-type gauche+ (χ1=−60°) rotamer and is oriented towards position 66.However, Cys66 adopts a trans rotamer (χ1=180°) and orients away fromposition 83. In the other two molecules, Cys66 adopts a gauche+(χ1=−60°) rotamer and is oriented towards position 83. However, position83 adopts a gauche− (χ1=+60°) rotamer and is oriented away from position66. Both of these alternative orientations is associated with noticeabledistortion of the local structure, but principally involving the gauche−rotamer of Cys83 and trans rotamer of Cys66 (see FIG. 12). With thegauche− rotamer of Cys83 the Sγ is oriented towards the main chainregion of residues 80 and 81, which shift their position ˜1.2 Å to avoidclose van der Waals contact. With the trans rotamer of Cys66 the Sγ isoriented towards the Tyr74 side chain Oη which shifts its position ˜1.4Å to avoid close van der Waals contact. There is substantially reducedstructural distortion associated with the alternative gauche+ rotamerfor position Cys66 and gauche+ rotamer for position Cys83. Thus, thestructural data show that for both Cys83 and Cys66 the rotamerorientations that are accommodated with the least structural distortionare compatible with disulfide bond formation (i.e., gauche+ in bothcases).

Ala66→Cys (oxidized): In contrast to the reduced form, the oxidized formof the Ala66→Cys mutant crystallized in the wild-type C222₁ space groupwith two molecules in the asymmetric unit. Both molecules in theasymmetric unit exhibit essentially identical structural features in theregion of the Cys66 mutation and show that the introduced Cys66 forms adisulfide bond with the wild-type Cys83 residue (FIG. 12, bottom panel).Each Cys residue adopts a gauche+ rotamer in forming a disulfide bond,and the local structural features are remarkably free of any apparentperturbation. The main chain atoms of all residues within 5.0 Å ofpositions 66 and 83 overlay with a root-mean-square deviation of 0.23 Å,essentially within the error of the X-ray data set. There is an ˜0.6 Åshift of the Cβ of Cys83 towards Cys66 in forming the disulfide, and thearomatic ring of adjacent Tyr55 correspondingly rotates ˜0.4 Å towardsCys83.

Example 1 Discussion

Cysteine is the second-least abundant amino acid in proteins (aftertryptophan), yet is among the most highly conserved in functionallyimportant sites involving catalysis, regulation, cofactor binding, andstability. See, e.g., Fomenko, D. E. et al. (2008), supra. The uniqueproperties of cysteine have their basis in the side chain Sγ sulfur atomparticipating in a variety of different functional roles, includingdisulfide bond formation, metal-binding, electron donation, hydrolysis,and redox-catalysis. However, some cysteine residues do not participatein these functional roles and exist instead as structural free-cysteineswithin the protein. These free cysteines are approximately evenlydistributed between interior and solvent exposed positions. See, e.g.,Petersen, M. T. N., (1999), supra. Free-cysteine residues within theinterior of a protein can effectively limit the protein's functionalhalf-life. These cysteines are potentially reactive thiols that aresubject to chemical modification should they become exposed, astransiently occurs in the dynamic equilibrium process of maintainingprotein structure. With regard to human FGF-1, structural data show thata buried free cysteine at position 83 participates as a rare H-bondacceptor, which suggests that the cysteine may be partially deprotonatedand therefore a reactive thiol. Chemical reactivity (e.g., disulfideformation) of buried free cysteines may present major structuraldifficulties for accommodation within the native protein interior, whichmay result in an irreversible unfolding pathway. As reported, mutationto eliminate these buried free cysteine residues may produce a notableincrease in functional half-life.

The absolutely conserved Cys83 in the FGF family of proteins includesthose members for whom this residue participates as a half-cystine(involving the adjacent Cys66) as well as those members for whom Cys83is a buried free cysteine (and adjacent position 66 is a non-cysteineresidue). Thus, while Cys83 is conserved among the FGF family ofproteins, Cys83 may have two distinctly different underlying roles: (1)a half-cystine which serves to stabilize protein structure; or (2) areactive buried free cysteine that may contribute to irreversibleunfolding and thereby regulate protein half-life. To better understandthe role of the conserved Cys83 in FGF-1 (a member of the Fgf1 subfamilywhere this position is a buried free cysteine), the effects of Ala, Ser,Thr, Val and Ile point mutations at position 83 upon stability andstructure are determined. Modeling studies indicate that unlike otheramino acids, this set of amino acids can substitute the wild-typecysteine residue without introducing unacceptably short van der Waalscontacts that would require gross structural adjustments (Gly is alsopossible, but can introduce a main chain entropic penalty). Thethermodynamic data (see FIG. 6) show that none of the amino acids inthis set can substitute for Cys83 without incurring a significant (i.e.,about 5.2 to about 12.6 kJ/mol) stability penalty (i.e., cysteine is theonly residue shown to be accommodated at position 83 without causingsignificant destabilization). Thus, there is a thermodynamic preferencefor a free cysteine at position 83 in FGF-1.

The X-ray data indicate that the structural environment surroundingCys83 is essentially rigid and unable to adapt to even small changesthat might otherwise accommodate alternative amino acids. Ser isisosteric with Cys and is typically considered a conservativesubstitution. However, the Ser oxygen atom has approximately 0.3 Åshorter van der Waals radius than sulfur. The hydrogen-bond betweenCys83 and the Asn80 main chain amide is 3.5 Å, and modeling an isostericserine into wild-type FGF-1 at position 83 results in a distance of 3.7Å between the serine Oγ and Asn80 amide. Thus, the structure mustcollapse 0.3-0.4 Å to maintain this H-bond interaction with a serinemutation. The X-ray structure shows little in the way of compensatingstructural collapse with the Cys83→Ala mutation to fill the void left bythe effective removal of the side chain Sγ, and therefore indicates thatthe surrounding structure of position 83 is relatively rigid. Thestructure of the Cys83→Ser mutation indicates a similarly limitedability to collapse. The average inter-atomic distance between theintroduced Ser83 Oγ and the Asn80 main chain amide for the twoindependent molecules in the asymmetric unit is 3.7±0.4 Å, in goodagreement with the modeling of a serine mutation in the wild-typestructure, and shows that the Asn80 main chain amide cannot effectivelyprovide an H-bond donor to the Ser at position 83. The hydrogen-bondingpartner that is observed in the Cys83→Ser mutant is the Oδ1 acceptor(2.7 Å distance) contributed by the reoriented Asn80 side chain. TheVal, Thr, and Ile mutations each exhibit some degree of close van derWaals contacts with the neighboring groups, necessitating structuraladjustments that are associated with a negative impact upon stability.The X-ray data therefore indicate that the local packing environment ofposition 83 is both relatively rigid, and optimized to accommodate afree cysteine at this position.

In addition to the structural interactions directly involving position83, analysis of the hydrogen-bonding details of adjacent positions showsthat these also contribute to the observed preference for cysteine atposition 83. Among the amino acids tested at this position, only withcysteine does the protein avoid unsatisfied H-bond interactionsinvolving main chain amides in the local turn structure (i.e., residues80 through 83). The Asn80 main chain amide is unsatisfied in theCys83→Ala, Thr, Val and Ile mutants, and the Glu82 main chain amide isunsatisfied in the Cys83→Ala mutant. On the other hand, both these mainchain amides are satisfied in their hydrogen-bonding requirement whencysteine is present at position 83 (see FIG. 11). Providinghydrogen-bonding partners for main chain amides in type 1 turns is knownto be important in stabilizing the turn structure. See, e.g., Lee J. etal. (2008), supra; de Alba, E. Et al., “Turn residue sequence determinesbeta-hairpin conformation in designed peptides,” Journal of the AmericanChemical Society 119:175-183 (1997); Santiveri, C. M. et al.,“Beta-hairpin folding and stability: molecular dynamics simulations ofdesigned peptides in aqueous solution,” Journal of Peptide Science10:546-565 (2004); and Wan, W.-Y. et al., “A natural grouping of motifswith an aspartate or asparagine residue forming two hydrogen bonds toresidues ahead in sequence: their occurrence at α-helical N termini andin other situations,” Journal of Molecular Biology 286:1633-1649 (1999),the entire contents and disclosures of which are hereby incorporated byreference. Thus, the destabilization associated with mutation of Cys83involves the contribution of both local and more extensivehydrogen-bonding interactions.

The structural changes in response to the Cys83→Ser mutation provideadditional insight into the unique properties of the cysteine residue atthis position. When a Ser is introduced, the protein responds to providean H-bond acceptor to the hydroxyl (i.e., the Oδ1 of Asn80). However,the sole H-bond partner for the wild-type cysteine is an H-bond donor(i.e., the nitrogen N of Asn80), even though the Asn80, side chain couldrotate to provide an acceptor (as observed for the Ser83 mutant). Cys83interacting as an H-bond acceptor, and not as a donor, suggests thatthis cysteine may be substantially deprotonated in the native structure.This is significant, not only because acting as an acceptor is therarest type of H-bond interaction observed for cysteines, but alsobecause it indicates that, in being deprotonated, Cys83 is a reactivethiol. See, e.g., Zhou, P. et al., “Geometric characteristics ofhydrogen bonds involving sulfur atoms in proteins,” Proteins (publishedonline 2008), the entire contents and disclosure of which are herebyincorporated by reference. The pK_(a) of an unperturbed cysteine is 8.4,and the X-ray data was collected at pH 7.5. Thus, it is not unreasonablethat the pK_(a) may be perturbed by the local electrostatic environment(possibly due to H-bond interaction with the Asn80 main chain amide orinteraction with the positive edge of the aromatic ring of adjacentTyr55) and that significant deprotonation of Cys83 occurs at pH 7.5.See, e.g., Britto, P. J. et al., “The local electrostatic environmentdetermines cysteine reactivity in tubulin,” Journal of BiologicalChemistry 277:29018-29027 (2002), the entire contents and disclosure ofwhich are hereby incorporated by reference. Thus, if position Cys83becomes solvent accessible (as during transient fluctuations in thefolding equilibrium), exposure of a reactive thiol would occur, andchemical modification of buried cysteines may contribute to anirreversible unfolding pathway. Therefore, position 83 in FGF-1 isoptimized to accept a free cysteine, and this buried free cysteine maynegatively regulate functional half-life.

The Ala66→Cys mutant tests the ability of the FGF-1 structure toaccommodate a disulfide bond with the conserved Cys83. Cys66 is presentat a corresponding position in six members of the FGF family within twoof the seven proposed archetype subfamilies (i.e., the Fgf8 subfamily,which includes FGF-8, 17 and 18, and the hFgf subfamily, which includesFGF-19, 21 and 23). See, e.g., Itoh, N., “The FGF families in humans,mice, Zebrafish: their evolutional processes and roles in development,metabolism, and disease,” Biological and Pharmaceutical Bulletin30:1819-1825, (2007), the entire contents and disclosure of which arehereby incorporated by reference.

Subsequent X-ray structure determinations proved that the Cys at aposition corresponding to position 83 of FGF-1 is a free thiol in FGF-1,-2, -4, -7, -9, -10, and -12. See, e.g., Blaber, M. et al., (1996),supra; Zhang, J. et al., “Three-dimensional structure of human basicfibroblast growth factor, a structural homolog of interleukin 1B,”Proceedings of the National Academy of Science USA 88:3446-3450 (1991);Bellosta, P. et al., “Identification of receptor and heparin bindingsites in fibroblast growth factor 4 by structure-based mutagenesis,”Molecular and Cellular Biology 21:5946-5957 (2001); Ye, S. et al.,“Structural basis for interaction of FGF-1, FGF-2, and FGF-7 withdifferent heparan sulfate motifs,” Biochemistry 40:14429-14439 (2001);Plotnikov, A. N. et al., “Crystal structure of fibroblast growth factor9 reveals regions implicated in dimerization and autoinhibition,”Journal of Biological Chemistry 276:4322-4329 (2001); Yeh, B. K. et al.,“Structural basis by which alternative splicing confers specificity infibroblast growth factor receptors,” Proc Natl Acad Sci USA100:2266-2271 (2003); and Olsen, S. K. et al., “Fibroblast growth factor(FGF) homologous factors share structural but not functional homologywith FGFs,” J Biol Chem 278:34226-36 (2003), the entire contents anddisclosures of which are hereby incorporated by reference. However,FGF-8, -17, -18, -19, -21 and -23 each contain a cysteine residue atposition 66 (in the numbering scheme of FGF-1) that lies adjacent toposition 83. The crystal structures of FGF-8, 19, and 23 have beensolved, and each shows a disulfide bond forms between Cys83 and theadjacent Cys66. See, e.g., Olsen, S. K. et al., “Structural basis bywhich alternative splicing modulates the organizer activity of FGF8 inthe brain,” Genes Dev. 20:185-198 (2006); Harmer, N. J. Et al., “Thecrystal structure of fibroblast growth factor (FGF) 19 reveals novelfeatures of the FGF family and offers a structural basis for its unusualreceptor affinity,” Biochemistry 43:629-640 (2004); and Goetz, R. etal., “Molecular insights into the klotho-dependent, endocrine mode ofaction of fibroblast growth factor 19 subfamily members,” Mol Cell Biol27:3417-3428 (2007), the entire contents and disclosures of which arehereby incorporated by reference.

The introduction of a cysteine mutation at adjacent position 66 resultsin a form of FGF-1 that is less stable than wild-type under reducingconditions, but substantially more stable than wild-type when exposed tooxidizing conditions with concomitant formation of an intra-moleculardisulfide bond. Under reducing conditions, the Ala66→Cys mutant in FGF-1is 5.1 kJ/mol less stable than the wild-type protein (see FIG. 6), andthe X-ray data shows some distortion of the local structure with both ofthe alternative conformations being observed for the pair of cysteinesat positions 66 and 83 (see FIG. 12). The purified Ala66→Cys proteinresponds to increased incubation under oxidative conditions by forming afaster-migrating band on SDS-PAGE, and an increase in stability of 13.6kJ/mol compared to the reduced mutant form (and 10.4 kJ/mol relative tothe wild-type protein). Thus, the SDS-PAGE results indicate theformation of a specific intra-molecular disulfide (with no evidence forformation of any inter-molecular disulfide bond). In the wild-type FGF-1(and reduced Ala66→Cys mutant) crystal structure, the Cβ-Cβ distancebetween position 66 and Cys83 is 4.3 Å, whereas the only other availablethiols in FGF-1 are Cys16 and Cys117, whose Cβ-Cβ distance to position66 are 18.0 Å and 12.0 Å, respectively. Thus, formation of a cystinebetween positions 66 and 83 in response to oxidation of the Ala66→CysFGF-1 mutant is consistent with the thermodynamic and structural dataprovided herein.

In the crystal structure of the reduced form of the Ala66→Cys mutant,two rotamer orientations for each cysteine at positions 66 and 83 areidentified, where the gauche+ rotamer in each case is necessary fordisulfide bond formation. gauche+ is the natural rotamer for thewild-type Cys83. The potential S-S distance when both cysteine rotamersare gauche+ is 2.20 Å, optimal for forming a disulfide bond. However,the Cβ-Sγ-Sγ-Cβ torsion angle is ˜0°, whereas a value of ˜90° iscanonical. See, e.g., Bhattacharyya, R. et al., “Disulfide bonds, theirstereospecific environment and conservation in protein structures,”Protein Eng Des Sel 17:795-808 (2004), the entire contents anddisclosure of which are hereby incorporated by reference. Thus,adjustments in χ1 or the Cα-Cβ bond vector (via adjustment in main chainϕ, ψ angles) for either, or both, residue positions appears necessary topromote disulfide bond formation. The isothermal equilibrium stabilitydata provided herein indicate that the strain introduced by achievingsuch an adjustment is more than offset by the entropically-based gain instability provided by formation of the disulfide bond.

The X-ray structure of the oxidized form of Ala66→Cys mutant showsformation of a disulfide bond between residues Cys66 and Cys83 (bothmolecules in the asymmetric unit are essentially identical in thisregard). The resulting Cβ-Sγ-Sγ-Cβ torsion angle is observed to be acanonical 90°, accomplished principally through a Cα-Cβ vectoradjustment involving position 83; other than this, the structuralperturbation in response to the introduction of the disulfide bond isnegligible. Thus, the wild-type structure readily accommodates theAla66→Cys mutant in a rotamer that favors disulfide bond formation withadjacent Cys83, and Cys83 in the wild-type structure appearsappropriately positioned (and also potentially deprotonated) to form adisulfide bond with an introduced Cys66. Formation of a disulfidebetween Cys 83 and Cys66 stabilizes the native structure by ˜10 kJ/mol(FIG. 6). The oxidized form of Ala66→Cys exhibits a more than 10 foldincrease in mitogenic activity and functional half-life in the absenceof heparin (FIG. 17, FIG. 22, FIG. 24), which unambiguously confirmsthat a newly formed disulfide bond in the Ala66→Cys mutant providesfavorable impact on functional stability of the protein.

A disulfide bond between cysteines at positions corresponding topositions 66 and 83 of human FGF-1 is present in two of the sevenarchetype subfamilies. As shown herein, wild-type FGF-1 is less stablethan the Cys66 mutant with the disulfide bond. However, low thermalstability of FGF-1 and FGF-2 may be essential to their non-traditionalsecretion mechanism. See, e.g., Florkiewicz, R. Z. et al., “Quantitativeexport of FGF-2 occurs through an alternative, energy-dependent,non-ER/Golgi pathway,” J Cell Physiol 162:388-99 (1995); Jackson, A. etal., “Heat shock induces the release of fibroblast growth factor 1 fromNIH 3T3 cells,” Proc Natl Acad Sci USA 89:10691-5 (1992); and Mach, H.et al., “Interaction of partially structured states of acidic fibroblastgrowth factor with phospholipid membranes,” Biochemistry 34:9913-9920(1995), the entire contents and disclosures of which are herebyincorporated by reference. As a potent mitogen, limiting functionalhalf-life may be important, and modulation of reactive thiols in FGF-1may influence the protein half-life by two-orders of magnitude. Bycontrast, the hFgf family, which does contain a cystine at positionscorresponding to positions 66 and 83, is unique in that these moleculesfunction in an endocrine fashion distal to the cells that secrete themwhen enhanced stability (as well as increased functional half-life) maybe more important. Thus, while Cys83 is absolutely conserved in the Fgffamily, the underlying basis for the selective pressure may differbetween the family members and involves differential issues of stabilityor regulation of functional half-life. The results with FGF-1 suggestthat other members of the Fgf family with a cysteine at position 83 maysimilarly be stabilized by a cysteine mutation at position 66.

Example 2 Interaction Between Thermostability and Buried Free Cysteinesin Regulating the Functional Half-Life of FGF-1

In this example, the relationship between protein stability and buriedfree cysteines in influencing the functional half-life of FGF-1 isexamined. Mutations that eliminate free cysteine residues are combinedwith protein stabilizing mutations to determine whether these mutationsmay have a combined, cooperative, and/or synergistic effect on proteinstability.

Materials & Methods

Mutagenesis, expression, and purification of recombinant proteins:Experiments in this example utilize a synthetic gene for the 140 aminoacid form of human FGF-1 containing an additional amino-terminal six Histag (SEQ ID NO: 14) as previously described. See, e.g., Ortega, S. etal. (1991), supra Gimenez-Gallego, G. et al. (1986), supra; Linemeyer,D. L. et al. (1990), supra; and Blaber, M. et al. (1996), supra. TheQuikChange™ site directed mutagenesis protocol (Stratagene) is used tointroduce mutations, and were confirmed by nucleic acid sequenceanalysis (Biomolecular Analysis Synthesis and Sequencing Laboratory,Florida State University). Expression and purification may followpreviously published procedures. See, e.g., Brych, S. R. et al. (2001),supra. Purified protein was exchanged into 50 mM NaPi, 0.1 M NaCl, 10 mM(NH₄)₂SO₄, 2 mM DTT, pH 7.5 (“crystallization buffer”) forcrystallization studies or 20 mM N-(2-acetamido)iminodiacetic acid(ADA), 0.1 M NaCl, 2 mM DTT, pH 6.6 (“ADA buffer”) for biophysicalstudies. The yield of most of the mutant proteins was 20-40 mg/L. Inthis example, an extinction coefficient of E_(280 nm) (0.1%, 1 cm)=1.26is used to determine protein concentration for wild-type and mutantproteins with the exception of those mutations involving Trpsubstitutions. See, e.g., Zazo, M. et al., “High-level synthesis inEscherichia coli of a shortened and full-length human acidic fibroblastgrowth factor and purification in a form stable in aqueous solutions”Gene 113:231-238 (1992); and Tsai, P. K. et al., “Formulation design ofacidic fibroblast growth factor,” Pharmaceutical Research 10:649-659(1993), the entire contents and disclosures of which are herebyincorporated by reference. Due to the addition of a novel Trpfluorophore in these proteins, their extinction coefficients aredetermined by densitometry analysis of Coomassie Brilliant Blue stainedSDS PAGE of serial dilutions of purified mutant proteins normalized toconcentration standards of wild-type FGF-1 (data not shown). Theresulting E_(280 nm) (0.1%, 1 cm) values used in this example are:Leu44→Trp=1.41; Phe85→Trp=1.55; Phe132→Trp=1.58;Leu44→Phe+Phe132→Trp=1.58;Cys83→Thr+Cys117→Val+Leu44→Phe+Phe132→Trp=1.58.

Crystallization, X-ray data collection, and refinement: Purified proteinin crystallization buffer is concentrated to 9-13 mg/ml, and crystalsare grown using the hanging-drop vapor diffusion method. Crystalssuitable for diffraction grow in one week at room temperature with 1.0ml of reservoir solution containing 2.0-3.5 M sodium formate and 0.1-1.0M ammonium sulfate in crystallization buffer. Crystals are mounted usingHampton Research nylon mounted cryo-turns and frozen in a stream ofgaseous nitrogen at 100K. In this example, diffraction data arecollected using a Rigaku RU-H2R rotating anode X-ray source (Rigaku MSC)equipped with Osmic Blue confocal mirrors (MarUSA) and a Rigaku R-axisIle image plate detector. Diffraction data are indexed, integrated andscaled using the DENZO software package. See, e.g., Otwinowski, Z. etal. (1993), supra; and Otwinowski, Z. et al. (1997), supra. His-taggedwild type FGF-1 (PDB code: 1JQZ) is used as the search model inmolecular replacement for mutant structures with the Crystallography andNMR System software (CNS). See, e.g., Brunger, A. T. (1998), supra.Model building and visualization utilizes the O molecular graphicsprogram. See, e.g., Johnson, D. E. et al. (1991), supra. Structurerefinement utilizes the CNS software, with 5% of the data in thereflection files set aside for R_(free) calculations. Coordinates andstructure factors are deposited in the PDB (coordinate file accessionnumbers are listed in the table in FIG. 13). Cavities within thestructures are quantified using the Molecular Surfaces Package (MSP)software and a 1.2 Å radius probe. See, e.g., Connolly, M. L. (1993),supra. The choice of 1.2 Å for the probe radius is slightly larger thanthe radius of a methyl group (1.1 Å) and identifies cavities that are ofsignificance for possible aliphatic or aromatic point mutations.

Isothermal equilibrium denaturation: Isothermal equilibrium denaturationby guanidine HCl (GuHCl) is performed using either fluorescence orcircular dichroism as the spectroscopic probe as previously described.See, e.g., Kim, J. et al. (2003), supra. FGF-1 contains a single buriedtryptophan residue at position 107 which exhibits atypically greaterfluorescence quenching in the native state versus the denatured state,and this differential fluorescence is used to quantify the unfoldingprocess. Fluorescence data are collected on a Varian Eclipsefluorescence spectrophotometer which is equipped with a Peltiercontrolled-temperature regulator at 298 K and using a 1.0 cm path lengthcuvette. 5.0 μM protein samples are equilibrated in ADA buffer at 298 Kin 0.1 M increments of GuHCl. Triplicate scans are collected andaveraged, and buffer traces are collected, averaged, and subtracted fromthe protein scans. Scans are integrated to quantify the totalfluorescence as a function of denaturant concentration.

The Leu44→Trp, Phe85→Trp, Phe132→Trp, Leu44→Phe+Phe132→Trp, andCys83→Thr+Cys117→Val+Leu44→Phe+Phe132→Trp mutations introduce anadditional tryptophan residue in the protein. In each case, thisadditional tryptophan exhibits greater fluorescence quenching in thedenatured state, and when combined with the endogenous Trp107 atypicalfluorescence signal results in an overall fluorescence quenching profilethat offers little discrimination between native and denatured states.FGF-1 unfolding monitored by circular dichroism (CD) spectroscopyexhibits excellent agreement with results obtained by fluorescencespectroscopy and is a useful alternative spectroscopic probe in caseswhere fluorescence cannot be utilized. Therefore, the isothermalequilibrium denaturation profile for the above mutants is characterizedusing CD spectroscopy. 25 μM protein samples are equilibrated in ADAbuffer at 298K in 0.1 M increments of GuHCl. In this example, CD dataare collected on a Jasco810 CD spectrophotometer (Jasco Inc) which isequipped with a Peltier controlled-temperature regulator at 298 K andusing a 1 mm path length cuvette. For each sample, triplicate scans arecollected and averaged, and buffer traces are collected, averaged, andsubtracted from the sample traces. The unfolding process is monitored byquantifying the change in CD signal at 227 nm with increasing GuHCl.See, e.g., Blaber, S. I. et al. (1999), supra. In this example, bothfluorescence and CD data are analyzed using the general purposenon-linear least-squares fitting program DataFit (Oakdale Engineering)implementing a six parameter, two-state model according to the followingequation (3):

$\begin{matrix}{F = \frac{F_{0\; N} + {S_{N}\lbrack D\rbrack} + {\left( {F_{0\; D} + \left( {S_{D}\lbrack D\rbrack} \right)} \right)e^{{- {({{\Delta \; G_{0}} + {m{\lbrack D\rbrack}}})}}/{RT}}}}{1 + e^{{- {({{\Delta \; G_{0}} + {m{\lbrack D\rbrack}}})}}/{RT}}}} & (3)\end{matrix}$

where [D] is the denaturant concentration, F_(0N) and F_(0D) are the 0Mdenaturant intercepts for the native and denatured state baselines,respectively, and S_(N) and S_(D) are the slopes of the native anddenatured state baselines, respectively. See, e.g., Eftink, M. R.(1994), supra. ΔG₀ and m describe the linear function of the unfoldingfree energy versus denaturant concentration. In this example, the effectof a given mutation upon the stability of the protein (ΔΔG) iscalculated by taking the difference between the C_(m) values forwild-type and mutant proteins and multiplying by the average of the mvalues, as described by Pace and Scholtz, according to the followingequation (4):

ΔΔG=(C _(m WT) −C _(m mutant))(m _(WT) +m _(mutant))/2   (4)

where a negative value indicates the mutation is stabilizing inrelationship to the wild type protein. See, e.g., Pace, C. N. (1997),supra.

Differential scanning calorimetry: In this example, DSC data arecollected on a VP-DSC microcalorimeter (MicroCal LLC) as previouslydescribed. See, e.g., Blaber, S. I. (1999), supra. Briefly, 40 μMprotein samples are equilibrated at 298 K in ADA buffer without DTT andin the presence of 0.7 M GuHCl. The samples are filtered and degassedfor 10 min prior to loading. A scan rate of 15 K/h is used and thesample was maintained at 30 psi during the calorimetric run. Proteinsamples are loaded, and data are collected without interruption ofrepeated thermal cycles. At least three independent protein scans arecollected and averaged, the average of the buffer scans is subtracted,and the resulting scan is normalized to the molar protein concentration.The resulting molar heat capacity profiles are analyzed using the DSCfitsoftware package. See, e.g., Grek, S. B. et al., “An efficient,flexible-model program for the analysis of differential scanningcalorimetry protein denaturation data,” Protein and Peptide Letters8:429-436 (2001), the entire contents and disclosure of which are herebyincorporated by reference.

Mitogenic activity and functional half-life in unconditioned medium:Purified protein is equilibrated in 0.14 M NaCl, 5.1 mM KCl, 0.7 mMNa₂HPO₄, 24.8 mM Tris base, pH 7.4 (“TBS buffer”), and the mitogenicactivity is evaluated by a cultured fibroblast proliferation assay aspreviously described. See, e.g., Dubey, V. K. et al. (2007), supra.Briefly, NIH 3T3 fibroblasts are plated in Dulbecco's modified Eagle'smedium (DMEM) (Gibco) supplemented with 0.5% (v/v) newborn calf serum(NCS) (Sigma) for 48 h at 37° C. with 5% (v/v) CO₂. The quiescentserum-starved cells are stimulated with fresh medium supplemented withFGF-1 protein (0-10 μg/ml) and incubated for an additional 48 hours.After this incubation period, the cells are counted using ahemacytometer (Hausser Scientific partnership). Experiments areperformed in quadruplicate, and cell densities are averaged. The proteinconcentration yielding one-half maximal cell density (EC₅₀ value) isused for quantitative comparison of mitogenicity. To evaluate the effectof exogenous heparin on mitogenic potency, 10 U/ml of heparin sodiumsalt (Sigma) is optionally added to the protein prior to cellstimulation.

For functional half-life studies, the wild-type and mutant FGF-1proteins are pre-incubated in unconditioned DMEM at 37° C. for varioustime periods (spanning 0-72 hr depending on the mutant) before beingused to stimulate the 3T3 fibroblast mitogenic response as describedabove. Although the mitogenic assay spans 48 hours, the stimulation ofFGF receptor in the initial minutes after FGF-1 addition may principallydictate the magnitude of the mitogenic response. Thus, evencomparatively short pre-incubation periods (i.e., less than 1 hour) maybe quantified for loss of functional activity. See, e.g., Ortega, S. etal. (1991), supra.

Resistance to thiol reactivity, aggregation and trypsin proteolysis inTBS: Wild-type and mutant proteins at a concentration of 0.25 mg/ml areincubated at 37° C. in TBS buffer and evaluated for disulfide bondformation and aggregation. Samples taken at time points of 0, 24, and 48hours are centrifuged at 10,000×g for 5 min, and the soluble fractionmixed with SDS sample buffer (both with and without 5% BME) resolved on16.5% Tricine SDS-PAGE and visualized with Coomassie Brilliant Bluestaining. The stained gels are scanned, and the amount of solublemonomeric protein and disulfide-linked multimers is quantified usingUN-SCAN-IT densitometry software (Silk Scientific).

Wild-type and mutant proteins are incubated with trypsin (Promega)(200:1 molar ratio, respectively) in TBS buffer at 37° C. to evaluateresistance to proteolysis. Time points are taken at 0, 5, 15 and 30 minand resolved on 16.5% Tricine SDS-PAGE visualized with CoomassieBrilliant Blue staining. The stained gels are scanned, and the amount ofintact protein is quantified using UN-SCAN-IT densitometry software(Silk Scientific).

Results

Mutant protein purification: Mutant proteins are expressed and purifiedto apparent homogeneity and with a yield similar to that of wild-typeprotein (20-40 mg/L).

X-ray structure determination: Diffraction-quality crystals are obtainedfor the Leu44→Trp, Phe85→Trp, Phe132→Trp, Val31→Ile, Cys117→Ile pointmutations, the Leu44→Phe+Phe132→Trp double mutant, and theLeu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp quadruple mutant. In thisexample, each of these mutant proteins crystallizes in the wild-typeorthorhombic C222₁ space group with two molecules in the asymmetricunit, and yielded 1.95-2.0 Å resolution data sets in each case. Thecrystal structures are refined to acceptable crystallographic residualsand stereochemistry (see table in FIG. 13). A brief description of eachrefined structure follows. However, in presenting the results adescription of the packing defects (i.e., cavities) within the core ofthe wild-type protein is necessary. The wild-type FGF-1 protein (1JQZ,molecule A) contains eight cavities detectable using a 1.2 Å radiusprobe. These cavities are identified by number (“cav1” through “cav8”)and details of their volume and location are provided in FIG. 18.

Summaries of individual X-ray structures is provided in the followingparagraphs:

Leu44→Trp: The mutant Trp side chain at position 44 is adopted with aχ1=−56° (similar to the wild-type Leu44 χ1=−44°) and χ2=90° (whichdiffers from the wild-type Leu44 χ2=165°) (FIG. 19A). Cav4 lies adjacentto the side chain of position 44, and the CZ2 atom of the mutant indolering occupies this region and effectively fills this cavity. The mutantTrp, however, introduces a close contact with the adjacent Ile sidechain at position 25, which responds by rotating from a gauche+ to transrotamer. In this orientation, the Ile25 Cδ1 atom occupies the adjacentcav6. This reorientation of the Ile25 side chain to accommodate themutant Trp also involves a 1.0 Å shift of the Ile25 main chain Cα awayfrom position 44, leading to an apparent increase in the Ile25N-His41Ointer-chain H-bond distance from 3.1 Å to 3.3 Å. The nitrogen in theindole ring of the mutant Trp H-bonds with the main chain carbonyl ofresidue Leu23 and is achieved with minimal structural perturbation.

Phe85→Trp: The mutant Trp side chain at position 85 is adopted with aχ1=−61° (essentially identical to the wild-type Phe85 χ1=−65°) andχ2=95° (identical to the wild type Phe) (FIG. 19B). Cav8 lies adjacentto the side chain of position 85 and the CZ3 atom of the mutant indolering occupies this region and substantially fills this cavity.Accommodation of the mutant Trp is associated with minimal perturbationof the surrounding structure. The nitrogen in the indole ring of themutant Trp H-bonds with the main chain carbonyl of residue Leu65 and isachieved with minimal structural perturbation.

Phe132→Trp: The mutant Trp side chain at position 132 is adopted with aχ1=−59° (similar to the wild-type Phe132 χ1=−68°) and χ2=85°(essentially identical to the wild-type Phe132 χ2=89°) (FIG. 19C). Twocavities are located adjacent to position 132: cav2 lies beneath thearomatic ring of Phe132 (and is the large central cavity characteristicof the β-trefoil architecture), and cav5 is adjacent to the introducedTrp CZ2 atom. See, e.g., Murzin, A. G. et al. (1992), supra. The mutantTrp side chain partially fills both of these cavities. Accommodation ofthe mutant indole ring is associated with minimal perturbation of thesurrounding structure. There is a slight rotation of the χ2 angle ofadjacent Leu111 as well as a slight repositioning of the main chaincarbonyl of adjacent residue Leu14, and both of these structuraladjustments are in a direction away from the mutant indole ring. Thenitrogen of the indole ring in the mutant Trp hydrogen bonds with themain chain carbonyl of residue Val109 and is achieved with minimalstructural perturbation.

Val31→Ile: The mutant Ile side chain at position 31 adopts a χ1=−55°(essentially overlaying the wild-type Val side chain at this position)and χ2=−60° (FIG. 19D). Cav6 lies adjacent to the introduced Ile Cδ1atom, but is only partially filled. However, in response to theintroduction of the mutant Ile Cδ1 at position 31, adjacent residueIle25 shifts in a direction away from position 31, such that the Cβ-Cβdistance between these neighboring groups increases from 5.6 Å to 6.1 Å.Thus, while the mutant Ile side chain partially fills an adjacentcavity, its accommodation is associated with positional adjustment ofneighboring side chains.

Cys117→Ile: The mutant Ile side chain at position 117 adopts a χ1=49°(essentially overlaying the mutant Ile Cγ2 atom onto the wild-type Sγatom) and a χ2=−175° (FIG. 19E). Cav5 is adjacent to position 117.However, the χ2 rotamer adopted by the mutant Ile side chain positionsits Cγ1 and Cδ1 atoms away from this cavity. The Ile mutation thereforehas no effect upon the size of adjacent cav5. Furthermore, thisorientation for the mutant Ile side chain positions the Cγ1 and Cδ1atoms outside of the core region, and these atoms become solventaccessible.

Leu44→Phe+Phe132→Trp: The structural effects of the Leu44→Phe pointmutant have been reported. See, Brych et al. (2001), supra. Briefly, theintroduction of the Phe aromatic ring essentially fills cav4, and theadjacent Ile25 side chain retains its rotamer orientation but shiftsposition away from Phe44 and fills cav6. Positions 44 and 132 are notadjacent packing neighbors and residues Leu14 and Leu23 are sandwichedbetween them. The structural effects of the Phe132→Trp point mutant isdescribed above, and the Leu44→Phe+Phe132→Trp double mutant can bedescribed as comprising the additive effects of the constituent pointmutations. In this regard, this combined double mutant effectively fillscav4, cav6 and partially fills cav5 (FIG. 20A).

Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp: The structural effects of theCys117→Val point mutant have been reported. See, e.g., Brych et al.(2003), supra. The Leu44→Phe/Phe132→Trp double mutant is describedabove. None of these four positions within the core region of theprotein are adjacent packing neighbors, and the effects of the combinedmutations may be described as comprising the additive effects of theconstituent point mutations. The Cys83→Thr and Cys117→Val mutations donot affect any of the 8 identified cavities in the structure, and thecavity-filling properties of this quadruple mutant are essentiallyidentical to that which is observed for the Leu44→Phe+Phe132→Trp doublemutant. These four mutations within the core region are accommodatedwith essentially no detectable change in the overall structuralbackbone. An overlay of the set of the main chain atoms within 5 Å frompositions 44, 83, 117 and 132 yields a root-mean-square deviation of0.23 Å (see FIG. 20B).

Isothermal equilibrium denaturation and differential scanningcalorimetry: The isothermal equilibrium data for the mutant proteins,exhibit excellent agreement in each case with a 2-state model. The Trpmutations at positions 44, 85 and 132 exhibit differential effects uponprotein stability. Leu44→Trp is destabilizing by 3.4 kJ/mol andPhe85→Trp is essentially neutral, whereas Phe132→Trp stabilizes theprotein by −1.6 kJ/mol (see table in FIG. 14). Both the Val31→Ile (4.0kJ/mol) and Cys117→Ile (1.5 kJ/mol) mutations destabilize the protein.The combined double mutant of Leu44→Phe+Phe132→Trp stabilize the proteinby −3.9 kJ/mol and therefore yields essentially additive effects uponstability in comparison to the constituent point mutations.

As reported, the Cys117→Val mutation, which effectively removes thisburied free cysteine, is only slightly destabilizing (1.2 kJ/mol), whilethe Cys83→Thr mutation substantially (5.2 kJ/mol) destabilizes theprotein. The combined Cys83→Thr+Cys117→Val double mutant, whicheliminates two of the three buried free cysteine residues in theprotein, destabilizes by 6.1 kJ/mol (see table in FIG. 14). This isessentially an additive effect of the constituent point mutations.Notably, combining the destabilizing Cys83→Thr+Cys117→Val double mutantwith the stabilizing Leu44→Phe+Phe132→Trp double mutant yields aquadruple mutant whose stability is indistinguishable from that of thewild-type protein (see table in FIG. 14). Thus, in theLeu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp quadruple mutant, two of thethree buried free-cysteines are eliminated while wild-type-equivalentstability is effectively maintained. The Lys12→Val+Cys83→Thr+Cys117→Valtriple mutant combines the destabilizing double cysteine mutant with apoint mutation (Lys12→Val; located in a partially solvent-accessiblesurface position) that has been shown to stabilize the protein by −7.8kJ/mol (and fills adjacent cav1). See, e.g., Dubey V. K. (2007), supra.The resulting Lys12→Val+Cys83→Thr+Cys117→Val triple mutant exhibits astability (−1.9 kJ/mol) that is slightly better than wild-type FGF-1 andis essentially the sum of the individual point mutations. Thus, thiscombined mutant eliminates two of the three buried free-cysteineresidues but has improved stability relative to wild-type FGF-1.

Differential scanning calorimetry data was collected for the Leu44→Trp,Phe85→Trp, and Phe132→Trp point mutations, as well as theLeu44→Phe+Phe132→Trp double mutant (See table in FIG. 15). The ΔΔGvalues derived from differential scanning calorimetry measurements arein excellent agreement with the isothermal equilibrium denaturation data(See table in FIG. 14). The results show that both ΔH and ΔS increasefor the stabilizing mutations, and both decrease for the destabilizingPhe85→Trp. The ΔΔG values for these mutations positively correlate withΔΔH and negatively correlate with −T*ΔΔS in each case. Thus, theobserved changes in stability for these mutations reflect anenthalpy-driven process. The DSC data is consistent with theintroduction of favorable van der Waals interactions for the Leu44→Pheand Phe132→Trp mutants, but not the Phe85→Trp mutant. The DSC data alsoconfirm the isothermal equilibrium data in showing that the effects uponmelting temperature and ΔG for the Leu44→Phe+Phe132→Trp double mutantare essentially additive with respect to the constitutive pointmutations.

Mitogenic activity and functional half-life in unconditioned medium: Themitogenic response or activity of NIH 3T3 cells with wild-type,Cys117→Val (C117V), Cys83→Thr+Cys117→Val (C83T/C117V),Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp (C83T/C117V/L44F/F132W), andLys12→Val+Cys83→Thr+Cys117→Val (C83T/C117V/K12V) mutant proteins (in thepresence and absence of heparin sulfate) is shown in FIG. 21. Both thewild-type and Cys117→Val mutant proteins exhibit a marked decrease inmitogenic activity in the absence of 10 U/ml exogenously added heparin(see table in FIG. 16). However, the Cys83→Thr+Cys117→Val,Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp, andLys12→Val+Cys83→Thr+Cys117→Val mutant proteins exhibit substantialmitogenic potency, even in the absence of exogenously added heparin.

Furthermore, the mitogenic response or activity of wild-type FGF-1 andAla66→Cys (A66C) mutant FGF-1 protein in both the absence or presence ofheparin is shown in FIGS. 22A and 22B. The EC₅₀ value of wild-type FGF-1in the absence of heparin is 58.4 ng/ml, while the Ala66→Cys mutantexhibits an EC₅₀ value of 5.43 ng/ml. Thus, the Ala66→Cys mutantexhibits ˜10-fold increase in mitogenic activity relative to wild-typeFGF-1 in the absence of added heparin. In the presence of heparin thewild-type FGF-1 and Ala66→Cys mutant are essentially indistinguishable,with EC₅₀ values of 0.48 ng/ml and 0.36 ng/ml, respectively (see tablein FIG. 17). These results show that the Ala66→Cys mutant has enhancedmitogenic activity in the absence of added heparin.

The mitogenic half-life of wild-type, Cys117→Val, Cys83→Thr+Cys117→Val,Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp, andLys12→Val+Cys83→Thr+Cys117→Val mutant FGF-1 proteins in response topre-incubation in unconditioned DMEM is shown in FIG. 23. The wild-typeprotein displays a pre-incubation half-life of 1.0 hour. However, withthe inclusion of the Cys117→Val mutation the half-life increases to 9.4hours. Subsequent addition of the Cys83→Thr mutation increases thepre-incubation half-life to 14.9 hours. When this Cys117→Val+Cys83→Thrdouble mutant is modified further by the addition of either thestabilizing Leu44→Phe+Phe132→Trp double mutant or the stabilizingLys12→Val point mutant, the half-life increases further to 42.6 and 40.4hours, respectively (see table in FIG. 16).

Furthermore, the residual activity of wild-type FGF-1 and the Ala66→Cys(A66C) mutant as a function of pre-incubation period in DMEM/0.5% NCSmedium at 37° C. is shown in FIG. 24 and FIG. 17. Under these conditionswild-type FGF-1 exhibits a functional half-life of 1.0 hr; however, theAla66→Cys mutant exhibits ˜14-fold increase (yielding a functionalhalf-life of 14.2 hr) (see table in FIG. 17).

Resistance to thiol reactivity, aggregation and trypsin proteolysis inTBS: The wild-type protein, and to a lesser extend the Cys117→Valmutant, exhibited visible precipitation after 24 and 48 hours incubationat 37° C. in TBS. The wild-type protein exhibits a general reduction intotal soluble protein as a function of incubation time in TBS (see FIG.25A). Furthermore, the non-reduced samples indicate the formation ofhigher-molecular mass forms, consistent with disulfide-linked dimers andtrimers, as a function of time. The Cys117→Val mutant (see FIG. 25B)yields a slight improvement in recovery of soluble material as afunction of incubation time (sec reduced lanes in FIG. 25B), althoughthe presence of higher-mass disulfide-linked forms is evident (seenon-reduced lanes in FIG. 25B). The Cys83→Thr+Cys117→Val double mutantimproves upon both the recovery of soluble protein (see reduced lanes inFIG. 25C) and the majority of the soluble protein is present as amonomeric form (see non-reduced lanes in FIG. 25C). This mutant has asingle Cys residue at position 16. Thus, the higher-mass form visibleunder non-reducing conditions is consistent with formation of aninter-molecular Cys16-Cys16 disulfide bonded dimer (i.e., about 36 kDa).The Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp (FIG. 25D) andLys12→Val+Cys83→Thr+Cys117→Val (FIG. 25E) mutant proteins showimprovements in both recovery of soluble material and fraction ofmonomeric form in comparison to the Cys83→Thr+Cys117→Val mutant, withthe Lys12→Val+Cys83→Thr+Cys117→Val mutant yielding the greatest recoveryof soluble monomeric protein after incubation.

The resistance to trypsin digestion for the wild-type, Cys117→Val,Cys83→Thr+Cys117→Val, Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Trp, andLys12→Val+Cys83→Thr+Cys117→Val mutant proteins is shown in FIG. 26. Theassociated half-life of the intact protein is given in the table in FIG.16. The Lys12→Val+Cys83→Thr+Cys117→Val exhibits the greatest resistanceto trypsin digestion (with a half-life of 19.1 min under the conditionstested), while the Cys117→Val mutant exhibits the greatestsusceptibility to trypsin digestion (with a half-life of 9.1 min).

Example 2 Discussion

The in vitro characterization of the functional half-life of the FGF-1protein demonstrates an interactive relationship between the reactivityof buried free cysteine residues and the thermodynamic stability of theprotein. Core packing mutations are shown to increase thermostabilityand counteract the minor destabilization associated with removal of freecysteine residues, while elimination of the free cysteines avoidsirreversible formation of disulfide bonds that are incompatible with thenative structure of FGF-1. Thus, stabilizing core packing mutations maybe combined with mutations that eliminate buried free cysteines toproduce substantial gains in functional half-life without heparin, whilemaintaining wild-type surface features and solvent structure that avoidpotential immunogenicity.

The previously reported X-ray structure of wild-type FGF-1 shows thatthe three free cysteines (at positions 16, 83, and 117) are each buriedwithin the protein interior and are 11-19 Å distal to each other. See,e.g., Blaber, M. (1996), supra. Formation of inter- or intra-moleculardisulfide bonds therefore requires substantial structural rearrangement(as would occur with protein unfolding), and is incompatible with nativeprotein structure and function. The half-life study of wild-type FGF-1in unconditioned DMEM indicates a functional half-life of about 1.0hour. Although the related incubation studies in TBS are not directlycomparable on the same time scale (due principally to concentrationdifferences utilized in these assays) the TBS study identifies aphysical basis for the observed loss of function (i.e., the incubationstudy of wild-type FGF-1 in TBS demonstrates loss of soluble monomelicprotein as a function of time due to irreversible aggregation).Furthermore, the soluble material that is recovered shows the formationof higher-mass disulfide adducts.

The Cys117→Val mutant eliminates one of three buried free cysteineresidues in FGF-1 and is associated with an increase in functionalhalf-life in unconditioned DMEM from 1.0 to 9.4 hours. This point mutantis essentially neutral with regard to its effects upon thermostability.Thus, the observed increase in functional half-life is likely dueexclusively to the elimination of a buried reactive thiol. Theincubation of the Cys117→Val mutant in TBS is associated with a markedreduction in visible aggregation, and the gel assay shows an increase inrecovery of soluble protein (although disulfide adducts involving Cys16and Cys83 are clearly present). (See FIG. 25) Elimination of a secondburied reactive thiol at position Cys83, with the Cys83→Thr+Cys117→Valdouble mutant, increases the half-life in unconditioned DMEM to 14.9 hr.The TBS incubation of this mutant shows improved recovery of solublemonomelic protein, and the disulfide adduct is now limited tointer-molecular dimer formation involving the remaining thiol atposition Cys16. The Cys83→Thr+Cys117→Val mutant is destabilizingcompared to the Cys117→Val mutant, and so the observed increase inhalf-life of this double mutant is due to the elimination of the secondburied reactive thiol and not due to an increase in thermostability. TheCys117→Val and Cys83→Thr+Cys117→Val mutants show that the elimination ofburied free cysteines within the structure is associated with asubstantial and combinatorial increase in the in vitro half-life.Accessibility of buried thiols requires unfolding of the protein, anddisulfide bond formation is an irreversible pathway from the denatured,state. Such pathways shift the folding equilibrium (via Le Chatelier'sprinciple) in the direction of the denatured state.

Comparing the Cys83→Thr+Cys117→Val,Leu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Val andLys12→Val+Cys83→Thr+Cys117→Val mutant proteins provides an opportunityto evaluate the effects of increasing thermostability, respectively,under conditions where the number and type of buried reactive thiols isheld constant (in this case, to the single remaining Cys16 residue). Incomparison to the Cys83→Thr+Cys117→Val mutant, theLeu44→Phe+Cys83→Thr+Cys117→Val+Phe132→Val mutant stabilizes the proteinby −6.5 kJ/mol, and the Lys12→Val+Cys83→Thr+Cys117→Val mutant stabilizesthe protein by −7.8 kJ/mol. Both of these mutants increase thefunctional half-life in unconditioned DMEM in comparison to theCys83→Thr+Cys117→Val mutant by a factor of three (from 14.9 hr to 42.6and 40.4 hr, respectively). This increase in functional half-life istherefore due exclusively to the increase in thermostability, as nochanges to buried thiols have been made. The incubation in TBS showsthat this increase in thermostability is associated with a reduction inthe formation of disulfide-bonded dimer and a corresponding increase inthe soluble monomelic form of the protein (FIGS. 22C through 22E). Theseresults are consistent with the hypothesis that denaturation isnecessary for buried free cysteines to become available for disulfidebond formation, and increasing protein stability shifts the foldingequilibrium towards the native state, thereby limiting the availabilityof the buried thiol for reactivity.

The addition of heparin to FGF-1 is known to stabilize the protein andincrease its melting temperature by about 20° C. See, e.g., Copeland, R.A. et al. (1991), supra. The addition of heparin to wild-type FGF-1increases its potency in the 3T3 fibroblast mitogenic assay by almosttwo orders of magnitude (see table in FIG. 16). However, the resultsshow that a similar enhancement in mitogenic activity is achieved in theabsence of added heparin for those FGF-1 mutant proteins that includethe Cys83→Thr+Cys117→Val double mutation (see FIG. 21). Furthermore, theCys117→Val mutation alone provides some enhancement in activity in theabsence of added heparin (although not to the extent observed for thedouble Cys mutants or in comparison to wild-type FGF-1 in the presenceof heparin). These results indicate that one of the major effects of theheparin-induced stabilization of FGF-1 is to effectively curtail buriedthiol reactivity. Point mutations that substantially stabilize the FGF-1protein have been shown to increase the mitogenic potency in the absenceof added heparin (see, e.g., Dubey, V. K. et al. (2007), supra), and thepresent results suggest that this stability effect upon mitogenicactivity is due principally to the abolishment of buried thiolreactivity.

Wild-type FGF-1 exhibits relatively poor thermal stability and containsthree free cysteines within the solvent-excluded core region. Thepresent results demonstrate a functional connection between buried freecysteines and thermostability, such that mutations affecting theseproperties may modulate the functional half-life. The results show thatin spite of potentially destabilizing effects, if buried thiols areeliminated by mutation, a significant increase in functional half-lifeis possible. Conversely, if the protein realizes a substantial gain inthermostability (i.e., due to mutation), the contribution of buried freecysteines in limiting functional half-life would be significantlydiminished. Thus, the combination of relatively low thermal stabilityand buried free cysteine residues in FGF-1 may represent co-evolvedproperties that cooperate to effectively regulate functional half-life.On the other hand, these two properties might be intentionallymanipulated in protein design efforts to achieve a desired targetfunctional half-life. Mutants with enhanced stability and functionalhalf-life offer potential advantages over the wild-type protein.However, there may be therapeutic applications where rapid clearance ofthe applied FGF protein is desirable. In this case, mutants forms withreduced half-life can potentially be designed by loweringthermostability and introducing additional buried cysteine residues.

Other properties, including susceptibility to proteolytic degradation,may contribute to the observed 3T3 fibroblast mitogenic half-life of themutant FGF-1 proteins. For the set of mutants tested, the resistance totrypsin digestion directly correlates with the thermodynamic stabilityof the protein (sec tables in FIGS. 14 and 16). Thus, in addition tolimiting the accessibility of buried reactive thiols, increasingthermostability protects the FGF-1 protein from loss of function due toproteolytic degradation. If mutation of buried free cysteines lowersthermodynamic stability, it can increase susceptibility to proteolyticdegradation and thereby contribute to a decrease in functionalhalf-life. In the case of Cys83 in FGF-1, a detailed X-ray structure andthermodynamic study shows that the local structural environment isoptimized to accept a cysteine at this position, and substitution byother residues results in significant destabilization (with theleast-disruptive mutation being Cys83→Thr). Thus, combining mutationsthat eliminate buried free cysteine residues with mutations thatincrease thermostability may offset instability associated with cysteinemutations and may have a synergistic effect upon functional half-life.

The manipulation of thermostability and/or buried free thiols is ofparticular interest in the design of “second generation” proteinbiopharmaceuticals. However, the immunogenic potential as a consequenceof mutational change is an important consideration. Substitution ofburied free cysteines may be constructed with stabilizing secondarymutations, such that these mutations are solvent inaccessible andaccommodated with minimal perturbation of the overall wild-typestructure. Mutations in FGF-1 may be designed to eliminate buried thiolsand/or contribute to protein stability, but leave the protein's surfacefeatures, including the solvent structure, indistinguishable from thatof wild-type. In attempting to achieve this goal, mutations to fillcore-packing defects may be made to thereby stabilize the proteinwithout introducing changes to the surface structure. In evaluating atotal of six core mutations, two are shown to be successful (i.e.,Phe132→Trp, and a previously described Leu44→Phe mutation), and one“broke even” (i.e., Phe85→Trp) (see table in FIG. 14). The DSC dataindicates that a net gain in van der Waals interactions is realized bythe successful subset of aromatic side chain mutations (i.e., Phe132→Trpand Leu44→Phe), but that the others (e.g., Phe85→Trp) are accommodatedwith an actual loss of favorable van der Waals interactions. Thus, thedisruption of local van der Waals interactions to accommodate the largeraromatic mutant side chains may offset any gain from the additionalburied area.

The two successful core mutations are combined to provide about 4 kJ/molof increased thermostability that may be used to offset an equivalentdecrease incurred by the elimination of two of the three buried freecysteines (Cys83→Thr, Cys117→Val). Thus, the elimination of two buriedthiols may be accomplished while maintaining overall thermostability,resistance to proteolysis, and most notably, achieving an approximately40× increase in functional half-life that eliminates the need forexogenously added heparin to achieve full mitogenic potency. An overlayof the main chain atoms of this quadruple mutant with those of wild-typeFGF-1 from the X-ray structures yields a root-mean-square deviation of0.25 Å, essentially identical to a similar overlay involving only thosepositions within 5.0 Å of the sites of mutation, and equivalent to theestimated error of the mutant X-ray data set. Furthermore, a total of 52conserved solvent molecules distributed over the surface of thewild-type and quadruple mutant proteins are in essentially identicalpositions when comparing the two structures (FIG. 27). Thus, thedesigned mutations within the solvent-excluded region of the protein areincorporated without perturbing the wild-type surface features,including solvent structure, and consequently, the immunogenic potentialof this mutant may be correspondingly minimized. This successful designprinciple may be applicable to a broad range of globular proteins thatcontain a buried free cysteine residue and core-packing defects.

FIG. 28 shows the mutant protein C83T/C117V/L44F/F132W (SEQ ID NO: 13).

While the present invention has been disclosed with references tocertain embodiments, numerous modification, alterations, and changes tothe described embodiments are possible without departing from the sphereand scope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

That which is claimed is:
 1. A mutant fibroblast growth factor (FGF)protein having a polypeptide sequence that is at least 90% identical tothe polypeptide sequence of wild-type human FGF-2 protein (SEQ ID NO:4), wherein the alanine (Ala) at an amino acid position of the mutantFGF protein corresponding to amino acid position 84 of wild-type humanFGF-2 is replaced with cysteine (Cys).
 2. The mutant FGF protein ofclaim 1, wherein the mutant FGF protein has a polypeptide sequence thatis at least 95% identical to the polypeptide sequence of wild-type humanFGF-2 protein (SEQ ID NO: 4).
 3. The mutant FGF protein of claim 1,wherein the mutant FGF protein has the polypeptide sequence of SEQ IDNO:
 6. 4. The mutant FGF protein of claim 1, wherein the mutant FGFprotein binds specifically to at least one FGF receptor (FGFR) aridtriggers growth or proliferation of cultured fibroblast cells.
 5. Afunctional fragment of the mutant FGF protein of claim 1, wherein thefunctional fragment of the mutant FGF protein binds specifically to atleast one FGF receptor (FGFR) and triggers growth or proliferation offibroblasts, endothelial cells, chondrocytes, osteoblasts, myoblasts,smooth muscle cells, glial cells, or neuroblasts.
 6. A polynucleotidesequence encoding the mutant FGF protein of claim
 1. 7. A host cellcontaining the polynucleotide sequence of claim
 6. 8. The mutant FGFprotein of claim 1 in a pharmaceutical composition including apharmaceutically acceptable carrier.
 9. A method, comprising thefollowing steps: (a) identifying an individual having an ischemiccondition or disease; and (b) administering to the individual acomposition comprising the mutant FGF protein of claim
 1. 10. The methodof claim 9, wherein the ischemic condition or disease is coronary arterydisease or peripheral vascular disease.
 11. The method of claim 9,wherein the composition is administered locally at or near the site ofthe ischemic condition or disease within the body of the individual. 12.The method of claim 11, wherein the composition is administered locallyat or near a site within the body of the individual causing the ischemiccondition or disease.
 13. The method of claim 12, wherein thecomposition is administered locally at or near an occluded blood vessel.14. A method, comprising the following steps: (a) identifying anindividual having a wound or tissue damage; and (b) administering to theindividual a composition comprising the mutant FGF protein of claim 1.15. The method of claim 14, wherein the wound or tissue damage is due toa traumatic injury or an immunologic disorder.
 16. The method of claim15, wherein the wound or tissue damage is due to stroke, myocardialinfarction, acute transverse myelitis (ATM), idiopathic transversemyelitis (ITM), brachial plexus injury, spinal cord injury, orperipheral nerve injury.
 17. The method of claim 14, wherein thecomposition is administered locally at or near the site of the wound ortissue damage within the body of the individual.
 18. A mutant fibroblastgrowth factor (FGF) protein having a polypeptide sequence that is atleast 90% identical to the polypeptide sequence of wild-type human FGF-2protein (SEQ ID NO: 4), wherein the phenylalanine (Phe) at an amino acidposition of the mutant FGF protein corresponding to amino acid position148 of wild-type human FGF-2 is replaced with tryptophan (Trp).
 19. Themutant FGF protein of claim 18, wherein the mutant FGF protein has apolypeptide sequence that is at least 95% identical to the polypeptidesequence of wild-type human FGF-2 protein (SEQ ID NO: 4).
 20. The mutantFGF protein of claim 18, wherein the mutant FGF protein has thepolypeptide sequence of SEQ ID NO:
 8. 21. The mutant FGF protein ofclaim 18, wherein the mutant FGF protein binds specifically to a FGFreceptor (FGFR) and triggers growth or proliferation of culturedfibroblast cells.
 22. A functional fragment of the mutant FGF protein ofclaim 18, wherein the functional fragment of the mutant FGF proteinbinds specifically to at least one FGF receptor (FGFR) and triggersgrowth or proliferation of fibroblasts, endothelial cells, chondrocytes,osteoblasts, myoblasts, smooth muscle cells, glial cells, orneuroblasts.
 23. A polynucleotide sequence encoding the mutant FGFprotein of claim
 18. 24. A host cell containing the polynucleotidesequence of claim
 23. 25. The mutant FGF protein of claim 18 in apharmaceutical composition including a pharmaceutically acceptablecarrier.
 26. The mutant FGF protein of claim 18, wherein the alanine(Ala) at an amino acid position of the mutant FGF protein correspondingto amino acid position 84 of wild-type human FGF-2 is replaced withcysteine (Cys).
 27. The mutant FGF protein of claim 18, wherein themutant FGF protein has the polypeptide sequence of SEQ ID NO:
 10. 28. Amethod, comprising the following steps: (a) identifying an individualhaving an ischemic condition or disease; and (b) administering to theindividual a composition comprising the mutant FGF protein of claim 18.29. The method of claim 28, wherein the ischemic condition or disease iscoronary artery disease or peripheral vascular disease.
 30. The methodof claim 28, wherein the composition is administered locally at or nearthe site of the ischemic condition or disease within the body of theindividual.
 31. The method of claim 28, wherein the composition isadministered locally at or near a site within the body of the individualcausing the ischemic condition or disease.
 32. The method of claim 31,wherein the composition is administered locally at or near an occludedblood vessel.
 33. A method, comprising the following steps: (a)identifying an individual having a wound or tissue damage; and (b)administering to the individual a composition comprising the mutant FGFprotein of claim
 18. 34. The method of claim 33, wherein the wound ortissue damage is due to a traumatic injury or an immunologic disorder.35. The method of claim 34, wherein the wound or tissue damage is due tostroke, myocardial infarction, acute transverse myelitis (ATM),idiopathic transverse myelitis (ITM), brachial plexus injury, spinalcord injury, or peripheral nerve injury.
 36. The method of claim 33,wherein the composition is administered locally at or near the site ofthe wound or tissue damage within the body of the individual.